TECHNICAL FIELD
The present disclosure relates generally to cold-responsive nanomaterials having applications in at least the medical and pharmaceutical industries. More particularly, but not exclusively, the present disclosure relates to in-situ cryo-immune engineering for cancer immunotherapy.
BACKGROUND
The background description provided herein gives context for the present disclosure. Work of the presently named inventors, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art.
Cancer is the second leading cause of death globally and metastatic cancer is the major reason for cancer-related mortality. Harnessing the immune system to battle cancer via immunotherapy has emerged as a powerful and potentially revolutionizing strategy for treating cancer metastasis. Therapeutic advances with tumor antigen vaccines, chimeric antigen receptor (CAR) T cells, and other cancer immunotherapy strategies have shown promising success in both pre-clinical and clinical studies of hematological malignancies. However, their efficacy of destroying solid tumors in most cancer patients has been limited. The immunosuppressive (i.e., immunologically cold) tumor microenvironment (TME) including malignant cells, deactivated/compromised immune cells, and soluble factors, is a key factor that contributes to the poor clinical outcomes of immunotherapy of solid tumors.
Cryosurgery has been proposed as a promising strategy to modulate the TME in cancer treatment. Cryosurgery is done by cooling to cause ice formation (i.e., frostbite) in tumor, which leads to cryoinjury and cancer cell death inside the frozen tissue iceball. This may result in the release of tumor antigens and production of damage-associated molecular patterns (DAMPs). These molecules may provoke immunogenic cell death (ICD), shape an immune-active TME, and further stimulate dendritic cells (DCs), macrophages, as well as CD8+ cytotoxic T lymphocytes (CTLs) to execute antitumor immunotherapy. In addition, unlike high-temperature thermal therapy for which vascular stasis occurs during heating, there is a temporary (a few hours) reperfusion of the tumor immediately after thawing a frozen tumor iceball, which may allow efficient immune cell infiltration into the tumor after cryosurgery. However, cryosurgery alone is insufficient to stimulate a potent immunotherapeutic effect against cancer.
Cryosurgery has been used clinically and become increasingly popular for treating cancers, particularly for breast cancer. This is because it is minimally invasive with minimized cosmetic damage compared to conventional surgery with a scalpel, and the frozen tumor iceball can be conveniently monitored in real time using medical ultrasonography due to the hyperechoic nature of ice. Unfortunately, a temperature of −20° C. or below is required to ensure cell death while the temperature in the peripheral region of a frozen tumor iceball detectable by medical ultrasonography is above −20° C. and up to ˜−4° C. The latter is because biological tissues freeze gradually with the decrease of temperature starting from ˜−0.6° C. to below −20° C. during cryosurgery, and the extent of ice formation is insufficient for detection (based on the aforementioned hyperechoic property of ice) by medical ultrasonography until ˜−4° C. In other words, medical ultrasonography can neither detect ice formation above ˜−4° C. nor tell where the temperature is below −20° C. that is needed to ensure cancer cell death. This may lead to incomplete tumor destruction and cancer recurrence after cryosurgery. Therefore, combining cryosurgery with other cancer therapies has been explored to improve cancer killing in the peripheral region of a frozen tumor iceball for enhanced therapeutic outcome. However, despite the efficacy of destroying localized tumors with the combination therapies, its capability of effectively killing distant and metastatic tumors (i.e., the abscopal effect of cryoimmunotherapy) has not been reported in the literature. Furthermore, no work has been done to utilize nanotechnology (particularly the one that is responsive to the cold/freezing temperature during cryosurgery) for delivering immunotherapy to combine with cryosurgery for enhancing the efficacy and safety of cancer therapy.
Thus, there exists a need in the art for an in-situ cryo-immune engineering (ICIE) approach to turn the TME from immunologically cold into hot, by developing a type of cold-responsive nanoparticles (CRNPs) to co-deliver chemotherapy and immunotherapy agents for combining with cryosurgery.
SUMMARY
The present disclosure utilizes co-encapsulated chemotherapy and immunotherapy agents in CRNPs for release into the cytosol in response to the cold temperature in the periphery of a frozen tumor iceball, for combining with cryosurgery. This activates a potent cryoimmune response against not only primary tumors on which cryosurgery is applied, but also distant and rechallenged metastatic tumors without cryosurgery, with excellent in vivo safety.
However, the CRNPs only actively target cancer cells and the chemotherapy drug does not perform its function in the cytosol. The efficacy and safety of the novel cryoimmunotherapy strategy can be further improved by developing the CRNPs with the capability of actively targeting cancer cells and co-encapsulating chemotherapy and immunotherapy agents that both function in the cytosol for cold-triggered cytosol-specific delivery.
The following objects, features, advantages, aspects, and/or embodiments, are not exhaustive and do not limit the overall disclosure. No single embodiment need provide each and every object, feature, or advantage. Any of the objects, features, advantages, aspects, and/or embodiments disclosed herein can be integrated with one another, either in full or in part.
It is a primary object, feature, and/or advantage of the present disclosure to improve on or overcome the deficiencies in the art.
It is a further object, feature, and/or advantage of the present disclosure to develop a novel targeting CRNPs-potentiated cancer cryoimmunotherapy.
It is still yet a further object, feature, and/or advantage of the present disclosure to understand the mechanisms of the novel cryoimmunotherapy for destroying primary, distant, metastatic, and rechallenged tumors.
It is still yet a further object, feature, and/or advantage of the present disclosure to enhance cancer cell killing at the edge of a frozen tumor iceball with a high subzero temperature (from ˜−4° C. to −20° C.) that alone cannot kill cancer cells.
It is still yet a further object, feature, and/or advantage of the present disclosure to stimulate cryo-immunotherapy against both the primary tumor with the cryosurgery treatment and distant/metastatic tumors with no cryosurgery.
It is still yet a further object, feature, and/or advantage of the present disclosure to utilize ICIE to reverse the immunosuppressive TME.
It is still yet a further object, feature, and/or advantage of the present disclosure to generate potent tumor-specific immune responses against primary, distant, and metastatic tumors with no evident side effect. This can be critical as cancer metastasis is the major cause of most cancer-related mortality.
It is still yet a further object, feature, and/or advantage of the present disclosure to checking the expression of PD-L1 and DAMPs together with the percentage of M1 vs. M2 macrophages, myeloid derived suppressor cells (MDSCs), CD8+ T cells vs. regulatory T (Treg) cells, and mature vs. immature DCs in primary, distant, and metastatic tumors.
It is still yet a further object, feature, and/or advantage of the present disclosure to examine the effector memory T (TEM) cells and central memory T (TCM) cells in blood and lymphoid organs to understand the abscopal and immunization effects of the cryoimmunotherapy described herein, in comparison to various controls. The novel targeting CRNPs-potentiated cryoimmunotherapy described herein can be critical to combat the metastasis of breast cancer and many other cancers.
The method for engineering an immunologically hot tumor microenvironment (TME) with cold-responsive nanomaterials (CRNPs) for cancer immunotherapy disclosed herein can be used in a wide variety of applications. The CNRPs can be utilized to control release of various drugs (e.g.: paclitaxel and doxorubicin). The targeting CPT&siR-laden CRNPs can be combined with cryosurgery for killing two different types of BC cells (EO711 and 4T1) in vitro and in vivo.
It is preferred that the targeting CNRPs be easy to use, cost effective, and stable.
Other surgeries and techniques to release drugs can be practiced which utilize novel targeting CRNPs-potentiated cancer cryoimmunotherapy which accomplish some or all of the previously stated objectives.
The targeting CRNPs can be incorporated into systems or kits which accomplish some or all of the previously stated objectives.
According to some aspects of the present disclosure, a method for engineering an immunologically hot tumor microenvironment (TME) with cold-responsive nanomaterials (CRNPs) for cancer immunotherapy, the method comprising targeting cancerous cells with the CRNPs during cryosurgery; utilizing a synthesized series of polymers that have lower critical solution temperatures (LCSTs) below positive four degrees Celsius (4° C.) to control release of a drug into a cytosol of said cancerous cells; and inducing cold-triggered endo/lysosomal escape of small interfering RNA (siRNA or siR) into the cytosol. Optionally, the lower critical solution temperatures (LCSTs) are below zero degrees Celsius (0° C.), and preferably, below negative four degrees Celsius (−4° C.).
According to some additional aspects of the present disclosure, the drug can comprise chemotherapy and immunotherapy agents.
According to some additional aspects of the present disclosure, the method can further comprise rapidly releasing an anticancer drug while moving siRNA (siR) out of the endosomes into the cytosol.
According to some additional aspects of the present disclosure, the method can further comprise allowing dendritic cells (DCs) to mature. Maturation is caused, at least in part, by enhancing production of damage-associated molecular patterns (DAMPs) so as to provoke immunogenic cell death (ICD). The method can even further comprise promoting the expression of the DAMPs including HMGB1, CRT, HSP-70, and HSP-90.
According to some additional aspects of the present disclosure, the method can further comprise activating T cells. The T cells can be CD8+ cytotoxic T cells activated with bone marrow dendritic cells (BMDCs). Circulating T cells in the blood can exert direct and rapid cytotoxicity against any existing tumors.
According to some additional aspects of the present disclosure, the method can further comprise utilizing memory immune cells induced by combining CRNPs with freezing to kill the primary tumor and to destroy a distant tumor without freezing said distant tumor.
According to some additional aspects of the present disclosure, the polymers are poly N-isopropylacrylamide copolymerized with butyl acrylate and the change of their ratios for copolymerization yields polymers with different LCSTs.
According to some additional aspects of the present disclosure, the method can further comprise co-encapsulating irinotecan (CPT) and programmed death-ligand 1 (PD-L1) silencing siRNA (siR) using a double-emulsion method. A synergistic effect of these two agents is produced when combined with cold treatment. Without cold treatment, the combined effect is not synergistic.
According to some additional aspects of the present disclosure, the method can further comprise decorating a surface of the resultant CPT and siR-laden CRNPs (CPT&siR CRNPs) with chitosan (CS) through the use of chitosan-modified PF-127.
According to some additional aspects of the present disclosure, a concentration of the CPT is approximately 10.0 μg ml−1.
According to some additional aspects of the present disclosure, the method can further comprise incubating the tumor cells expressing the green fluorescent protein (GFP) with GFP silencing siRNA laden CRNPs to produce a gene silencing effect.
According to some additional aspects of the present disclosure, the method can further comprise injecting phosphate buffered saline (PBS) into the cancerous cells.
According to some additional aspects of the present disclosure, the method can further comprise combining cryosurgery with CPT& PD-L1 silencing siRNA CRNPs to induce a more potent antitumor immune response than one of the single treatment.
According to some additional aspects of the present disclosure, the method can further comprise manipulating a killing temperature of the cancerous cells by turning a cryoprobe on and off intermittently.
According to some additional aspects of the present disclosure, the method can further comprise attenuating a frequency of monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly6C+Ly6G−) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b+Ly6C−Ly6G+), pro-tumorigenic tumor associated macrophages (F4/80+CD206+CD86−), and regulatory T cells (Tregs, CD4+Foxp3+) that perform immunosuppressive activities in the tumor microenvironment (TME).
According to some other aspects of the present disclosure, cold-responsive nanomaterials (CNRPs) capable of targeting cancerous cells comprise irinotecan (CPT); and programmed death-ligand 1 (PD-L1) co-encapsulated with said CPT.
According to some additional aspects of the present disclosure, the CNRPs co-encapsulated with said CPT are laden with siR (CPT&siR CRNPs).
According to some additional aspects of the present disclosure, a surface of the CNRPs are decorated with chitosan (CS) through the use of chitosan-modified PF-127.
According to some additional aspects of the present disclosure, a concentration of the CPT is approximately 10.0 μg ml-1.
According to some additional aspects of the present disclosure, the CNRPs comprise memory immune cells that when combined with freezing can kill a primary tumor and can destroy a distant/metastatic tumor without freezing said distant tumor.
According to some other aspects of the present disclosure, a system for carrying out a cryosurgery comprises cold-responsive nanomaterials capable of targeting cancerous cells comprise a synthesized series of polymers that have lower critical solution temperatures (LCSTs) below positive four degrees Celsius (4° C.) to control release of a drug into a cytosol of said cancerous cells; and a drug that can be released into a cytosol of said cancerous cells. The drug can comprise chemotherapy and immunotherapy agents. The drug can be an anticancer drug that is rapidly released while moving siRNA (siR) into the cytosol after cold treatment. Optionally, the lower critical solution temperatures (LCSTs) are below zero degrees Celsius (0° C.), and preferably, below negative four degrees Celsius (−4° C.).
According to some additional aspects of the present disclosure, the system further comprises an area where dendritic cells (DCs) are allowed to mature. Maturation can be caused, at least in part, by an enhancement in the production of damage-associated molecular patterns (DAMPs) so as to provoke immunogenic cell death (ICD). The system can even further comprise promoters of the DAMPs, including HMGB1, CRT, HSP-70, and HSP-90.
According to some additional aspects of the present disclosure, the system further comprises T cells. The T cells can be CD8+ cytotoxic T cells activated with bone marrow dendritic cells (BMDCs). The system can further comprise a pump that circulates the T cells in the blood so as to exert direct and rapid cytotoxicity against any existing tumors.
According to some additional aspects of the present disclosure, the system further comprises memory immune cells induced by combining CRNPs with freezing to kill a primary tumor and to destroy a distant/metastatic tumor without freezing said distant tumor.
According to some additional aspects of the present disclosure, the polymers are poly N-isopropylacrylamide copolymerized with butyl acrylate and the change of their ratios for copolymerization yields polymers with different LCSTs.
According to some additional aspects of the present disclosure, the system further comprises green fluorescence protein (GFP) tumor cells incubated with GFP silencing siRNA loaded inside the CRNPs to produce a GFP gene silencing effect.
According to some additional aspects of the present disclosure, the system further comprises CPT& PD-L1 silencing siRNA CRNPs that are combined with cryosurgery to induce a more potent antitumor immune response than one of the single treatment.
According to some additional aspects of the present disclosure, the system further comprises a temperature control for manipulating a killing temperature of the cancerous cells by turning a cryoprobe on and off intermittently.
These and/or other objects, features, advantages, aspects, and/or embodiments will become apparent to those skilled in the art after reviewing the following brief and detailed descriptions of the drawings. The present disclosure encompasses (a) combinations of disclosed aspects and/or embodiments and/or (b) reasonable modifications not shown or described.
BRIEF DESCRIPTION OF THE DRAWINGS
Several embodiments in which the present disclosure can be practiced are illustrated and described in detail, wherein like reference characters represent like components throughout the several views. The drawings are presented for exemplary purposes and may not be to scale unless otherwise indicated.
FIGS. 1A-1D shows synthesis and characterization of cold-responsive pNIPAM-BA polymers. FIG. 1A shows a synthetic rout and proton-nuclear magnetic resonance (1H-NMR) spectra of pNIPAM-BA polymers with different NIPAM to BA ratios, showing successful synthesis of pNIPAM-BA polymers with typical bonds labelled as a-g. NIPAM: N-isopropylacrylamide, BA: butyl acrylate, AIBN: 2,20-Azobis(2-methylpropionitrile). FIG. 1B shows gel permeation chromatography (GPC) spectra of the synthesized pNIPAM-BA polymers with different NIPAM to BA ratios. The data of retention time (t) labelled on the figure was used to calculate the number-averaged molecular weight (Mn) of polymer based on a calibration curve for which the method is detailed in the Methods section. FIG. 1C shows a molecular weight (Mn), polydispersity index (POI), and number of each monomer (x for NIPAM and y for BA) in the different pNIPAM-BA polymers calculated from 1H-NMR and GPC spectra. FIG. 1D shows that a higher content (judged by the ratio of x to y) of NIPAM in the resultant pNIPAM-BA polymers leads to higher LCST regardless of the solvent/solution where they are dissolved, including deionized water (OW), cell culture medium (medium), saline, and phosphate buffer saline (PBS) at both pH 7.4 and 5.0. n=3. Two-way analysis of variance (ANOVA) with Tukey's multiple comparisons and correction was used for statistical analyses.
FIG. 2 shows a schematic illustration of the ICIE strategy for killing both primary and distant tumors. ICIE combines cryosurgery with intravenously injected (i.v.) irinotecan (or camptothecin/CPT) and PD-L1 siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting the immunogenic cell death (ICD) and reducing the expression of programmed cell death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immune-active). As a result, the CD8+T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAM-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and TEM: effective memory T cell.
FIGS. 3A-3E show CPT&siR CRNPs with optimized CPT loading show good stability and prevent siR from fast degradation. FIGS. 3A-3B show the loading capacity (LC, FIG. 3A) and encapsulation efficiency (EE, FIG. 3B) of CPT in CRNPs after feeding CPT at 1, 3, and 5% wt of the polymers during the preparation (n=3 independent experiments). FIG. 3C shows CPT&siR CRNPs display no significant changes in their size and size distribution (indicated by PDI) after incubating with PBS for 5 days (n=3 independent experiments). FIG. 3D shows free siR shows fast degradation (within five minutes (5 min) of incubation with PBS at twenty two degrees Celsius (22° C.)) as determined by the agarose gel electrophoresis. The black bands are due to the background of the 6× loading buffer. FIG. 3E shows CPT&siR CRNPs prevent siR from degradation after at least twenty four hours (24 h) of incubation with PBS at twenty two degrees Celsius (22° C.), indicated by the evident siR band (white) in agarose gel well. The experiments for FIGS. 3D-3E were repeated three times independently with similar results. Data are presented as mean±SD (FIGS. 3A-3C).
FIGS. 4A-4I show cold-responsiveness of CRNPs for enhanced endo/lysosomal escape of siRNA and gene silencing. FIG. 4A shows transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of CPT&siR CRNPs before and after cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min). FIG. 4B shows hydrodynamic size (diameter) distribution of the CPT&siR CRNPs before and after the cold treatment. FIG. 4C shows typical photographs and cryo-microscopy images of CPT&siR PLGA NPs that are not cold-responsive and CPT&siR CRNPs at different temperatures, showing the cooling-enhanced transparency of the aqueous suspension of the CRNPs due to their disassembly at cold temperature. Scale bar: 500 μm. FIG. 4D shows average grayscale intensity of the aqueous samples of CPT&siR PLGA NPs and CPT&siR CRNPs from their cryo-microscopy images taken at different temperatures to show the cold-responsiveness of the CPT&siR CRNPs. FIG. 4E shows confocal images (left 5 columns), fluorescence intensity distribution (column 6), and intensity distribution of Cy5-siR (right column) of EO771 cells after incubation with PBS, free CPT&Cy5-siR, CPT&Cy5-siR CRNPs, and CPT&Cy5-siR CRNPs+C for eight hours (8 h). “+C” indicates cold treatment at −4° C. for ten minutes (10 min) done after the eight hours (8 h) incubation. Blue: 4′,6-diamidino-2-phenylindole (DAPI bound to DNA in nuclei, excited at 405 nm that does not excite CPT fluorescence), red: cyanine 5 labeled siRNA (Cy5-siR), green: LysoTracker Green. FIGS. 4F-4G show expression of GFP fluorescence characterized by flow cytometry showing typical flow cytometry histograms (FIG. 4F) and the quantitative data (FIG. 4G) of GFP+ EO771 cells either with no treatment or after treated with free GFP-siR, GFP-siR CRNPs, GFP-siR CRNPs+C, and Sc-siR CRNPs+C (n=3 independent experiments). GFP: green fluorescence protein. Sc-siR: scrambled siRNA, “+C”: indicates cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min). FIGS. 4H-4I, Expression of PD-L1 analyzed by flow cytometry showing typical flow cytometry histograms (FIG. 4H) and the quantitative data (FIG. 4I) of EO771 cells after different treatments (n=3 independent experiments). Statistical analyses were done using one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test and correction. The experiments for FIGS. 4A-4E were repeated three times independently with similar results. Data are presented as mean±SD (FIGS. 4G, 4I).
FIGS. 5A-5D show CPT&siR CRNPs release more siR at negative four degrees Celsius (−4° C.), but not at twenty two degrees Celsius (22° C.) and thirty seven degrees Celsius (37° C.), as shown by the agarose gel electrophoresis. SA: sample. The experiments were repeated three times independently with similar results. FIG. 5B-5D show CPT&siR CRNPs show cold-triggered release of CPT and siR at pH 5.0 (acetate buffer), pH 6.5 (phosphate buffer), and pH 7.4 (phosphate buffer) (n=3 independent experiments). Cold treatment was performed by incubating the samples at negative four degrees Celsius (−4° C.) for ten minutes (10 min). Data are presented as mean±SD (FIGS. 5B-5D).
FIGS. 6A-6D show CPT&Cy5-siR CRNPs enhance cellular uptake of siR in EO771 cancer cells with negligible uptake in lymphocytes. FIG. 6A shows cellular uptake of free CPT&Cy5-siR and CPT&Cy5-siR CRNPs in GFP+ EO771 cells after incubation for eight hours (8 h). Green: green fluorescent protein (GFP), blue: 4′,6-diamidino-2-phenylindole (DAPI), and red: cyanine 5 (Cy5). FIGS. 6B-6C show a further determination of cellular uptake in GFP+ EO771 cells by flow cytometry (FIG. 6B) and the corresponding quantitative analysis (FIG. 6C) after incubating the cells with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs for eight hours (8 h) (n=3 independent experiments). FIG. 6D shows flow cytometry analysis of cellular uptake of free CPT&Cy5-siR and CPT&Cy5-siR CRNPs in normal spleen lymphocytes after incubation for eight hours (8 h). The experiments were repeated three times independently with similar results. One-way ANOVA with Tukey's multiple comparisons and correction was used for statistical analyses. Data are presented as mean±SD (FIG. 6C).
FIG. 7 shows CPT&Cy5-siR CRNPs with no NaCl induce minimal endo/lysosomal escape. Representative confocal images showing minimal endo/lysosomal escape of Cy-5-siR in EO771 cells treated with free CPT&Cy5-siR, CPT&Cy5-siR CRNPs (no NaCl), CPT&Cy5-siR CRNPs (no NaCl) +C. “+C” indicates the groups with cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min). NaCl: sodium chloride. The experiments were repeated three times independently with similar results.
FIG. 8 shows the treatment of CRNPs+C shows strong GFP silencing effect in GFP+ EO771 cells. Representative confocal images of GFP+ EO771 cells treated with free GFP-siR,
GFP-siR CRNPs, GFP-siR CRNPs+C and Sc-siR CRNPs+C. “+C” indicates the groups with cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min). Sc-siR: scrambled siR (as negative control). The experiments were repeated three times independently with similar results.
FIG. 9 shows cold treatment enhances the in vitro cancer cell killing effect of CPT&siR CRNPs. EO771 cells were treated with blank CRNPs, free CPT&siR, CPT&siR CRNPs, and CPT&siR CRNPs+C. “+C” indicates the groups with cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min) (n=6 independent experiments). Two-way ANOVA with Sidak's post-test and correction for multiple comparisons was used for statistical analyses. Data are presented as mean±SD.
FIG. 10 shows CPT&siR CRNPs+C induce more apoptosis in cancer cells. EO771 cells treated with PBS control, free CPT&siR, CPT&siR CRNPs, and CPT&siR CRNPs+C were stained with annexin V (for early and late apoptosis) and propidium iodide (PI, for apoptosis/necrosis) (n=3 independent experiments). “+C” indicates the groups with cold treatment at negative four degrees Celsius (−4° C.) for ten minutes (10 min). One-way ANOVA with Tukey's multiple comparisons and correction was used for statistical analyses. Data are presented as mean±SD.
FIG. 11 shows cryosurgery reinforces CPT&siR CRNPs in inducing T cell activation and tumor attacking via production of ICD. FIG. 11A shows expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at negative twenty degrees Celsius (−20° C.), negative four degrees Celsius (−4° C.), or thirty seven degrees Celsius (37° C.) for ten minutes (10 min) (n=3 independent experiments). FIG. 11B shows a schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8+ cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). FIGS. 11C-11D show typical flow cytometry plots (FIG. 11C) and quantitative data (FIG. 11D) of mature bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for twenty four hours (24 h) (n=3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at negative twenty degrees Celsius (−20° C.) for ten minutes (10 min). FIGS. 11E-11F, Quantitative data (e) and typical flow cytometry plots (f) of CD11c+Kb-SIINFEKL+ BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells (n=3 independent experiments). FIGS. 11G-11H typical flow cytometry histograms (FIG. 11G) and quantitative data (FIG. 11H) of CD8+ T cell proliferation after incubating them with the BMDCs activated by the EO771-OVA cells treated with aforementioned formulations (n=3 independent experiments). FIGS. 11I-11J show typical flow cytometry plots (FIG. 11I) and quantitative data (FIG. 11J) of activated CD8+ T cells following co-culture with the aforementioned BMDCs (n=3 independent experiments). FIG. 11K shows representative images showing the process of activated CD8+ T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. FIGS. 11L-11M show typical flow cytometry plots (FIG. 11L) and quantitative data (FIG. 11M) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations (n=3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey's multiple comparisons and correction. Data are presented as mean±SD (FIG. 11A, 11D-11E, 11H, 11J, 11M).
FIG. 12A-12B show promotion of the secretion of pro-inflammatory cytokines for boosting the antitumor immune response by CPT&siR CRNPs with cold treatment. FIGS. 12A-12B show production of IFN-γ (FIG. 12A) and TNF-α (FIG. 12B) measured by ELISA after co-culturing T cells with bone marrow dendritic cells (BMDCs, CD11c+CD86+) matured by EO771-OVA cells for twenty four hours (24 h) (n=3 independent experiments). The EO771-OVA cells were pretreated with PBS, free CPT&siR, CPT&siR CRNPs, or CPT&siR CRNPs+C. “+C” represents cold treatment at negative twenty degrees Celsius (−20° C.) for ten minutes (10 min). Statistical analyses were performed using one-way ANOVA with Tukey's multiple comparisons and correction. Data are presented as mean±SD (FIGS. 12A-12B).
FIGS. 13A-13C show a synergistic effect of CPT and siR co-delivered in CPT&siR CRNPs with cold treatment. FIGS. 13A-13B show typical flow cytometry plots (FIG. 13A) and quantitative data (FIG. 13B) of dead EO771-OVA cells after T cell attacking for experiment without cold treatment. T cells were activated by BMDCs co-cultured with EO771-OVA cells with treatment of CPT CRNPs, siR CRNPs, or CPT&siR CRNPs (n=3 independent experiments). PI: propidium iodide. The group labeled with a dotted box represents the sum value (i.e., simple additive effect on cancer cell death) of CPT CRNPs and siR CRNPs. FIGS. 13C-13D show typical flow cytometry plots (FIG. 13C) and quantitative data (FIG. 13D) of dead EO771-OVA cells after T cell attacking for experiment with cold treatment (+C) (n=3 independent experiments). T cells were activated by BMDCs co-cultured with EO771-OVA cells with treatment of CPT CRNPs+C, siR CRNPs+C, or CPT&siR CRNPs+C. The group labeled with dotted box represents the sum value (i.e., simple additive effect on cancer cell death) of CPT CRNPs+C and siR CRNPs+C. Statistical analyses were done using one-way ANOVA with Tukey's multiple comparisons and correction. Data are presented as mean±SD (FIG. 13B, 13D).
FIGS. 14A-14B show CRNPs show high accumulation in orthotopic breast tumors in vivo and excellent blood compatibility. FIGS. 14A-14B show whole animal images (FIG. 14A) showing the in vivo distribution of CPT&Cy5-siR CRNPs and the corresponding quantitative data (FIG. 14B) of the fluorescence intensity of CPT&Cy5-siR CRNPs in E0771 tumors from IVIS imaging at different time points after intravenous injection of the CRNPs. PBS and free CPT&Cy5-siR were investigated for comparison (n=3 mice). The blue arrows indicate the locations of tumors. FIG. 14C shows representative images acquired by IVIS imaging showing the Cy5 fluorescence intensity in blood drawn at various time points from mice after injection with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs (at the same Cy5-siR dose as free CPT&Cy5-siR) (n=3 mice). FIGS. 14D-14E, Typical IVIS images (FIG. 14D) and the corresponding quantitative data (FIG. 14E) showing the Cy5 fluorescence in different organs including tumors collected from mice injected via tail vein with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs and sacrificed at twenty four hours (24 h) after the injections (n=3 mice). FIG. 14F shows hemolysis assay of CPT&siR CRNPs at different NP concentrations ranging over 25-800 μg ml−1 (n=3 independent experiments). Deionized water (DW) and PBS were used as the positive (˜100% hemolysis) and negative (˜0% hemolysis) controls, respectively. Insets are the representative SEM images of red blood cells incubated with PBS and CPT&siR CRNPs at a concentration of 800 μg ml−1. Scale bar: 2 μm. ns: no significance. Statistical analyses were done using one-way ANOVA with Tukey's multiple comparisons and correction. The experiments for FIGS. 14C-14D were repeated three times independently (n=3 mice) with similar results. Data are presented as mean±SD (FIG. 14B, 14E-F).
FIG. 15 shows CPT&Cy5-siR CRNPs extend the blood circulation time after injection. Blood was collected at zero hours (0 h), four hours (4 h), eight hours (8 h), twelve hours (12), and twenty four hours (24 h) after intravenously injecting the mice with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs (n=3 mice). One-way ANOVA with Tukey's multiple comparisons and correction was used for statistical analyses.
FIGS. 16A-16J show ICIE reverses immunologically “cold” TME into a “hot” one. FIG. 16A shows a schematic illustration of the experimental design for mice receiving only intravenous injection of various formulations every three (3) days with no cryosurgery. For these mice, the tumors on the left and right sides are indicated as L and R tumors, respectively. FIG. 16B shows a schematic illustration of the in vivo experimental design for mice receiving both intravenous injection of various formulations and cryosurgery done only on the primary (Prim) tumors on the left side. The tumors that undergo no freezing on the right side of these mice are indicated as distant (Dist) tumors. Cryosurgery was performed at eight hours (8 h) after the first injection of the formulations. FIG. 16C shows a typical photograph of the tumor area of a mouse during cryosurgery. Thermocouple 1 (TC1) and TC2 are for monitoring the temperature in tumor outer boundary and center (next to the cryoprobe), respectively. FIG. 16D shows typical thermal histories recorded at the tumor boundary and center during the cryosurgical procedure. FIG. 16E shows representative FLIR infrared images and photographs of mice showing the tumor temperature and appearance at three different time points before (0 s), during (300 s), and after (630 s) cryosurgery. FIG. 16F shows representative immunofluorescence images showing the expression of HMGB1, CRT, HSP-70, and HSP-90 in primary tumors treated by the indicated formulations in combination with cryosurgery. FIGS. 16G-16J show quantitative flow cytometry data on the ratio of tumor associated macrophages M1 (F4/80+CD206−CD86+) to M2 (F4/80+CD206+CD86−) (FIG. 16G), percentage of monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly6C+Ly6G−, FIG. 16H), percentage of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b+Ly6C−Ly6G+−, FIG. 16I), and percentage of regulatory T (Treg) cells (CD4+Foxp3+, FIG. 16J) in primary (Prim) tumors of mice with both cryosurgery and injection of one of the formulations and in L (left side) tumors of mice injected with the different formations only and with no cryosurgery (n=3 mice). Statistical analyses were done using two-way ANOVA with Sidak's post-test and correction for multiple comparisons. The experiments for FIGS. 16E-F were repeated three times independently (n=3 mice) with similar results. Data are presented as mean±SD (FIGS. 16G-16J).
FIGS. 17A-17B show ICIE enhances the in vivo PD-L1 silencing efficacy. FIG. 17A shows representative western blot results showing that ICIE induces better PD-L1 silencing in vivo among all primary tumors of mice receiving cryosurgery and injections of one of the various formulations including PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs. FIG. 17B shows PD-L1 silencing in left tumors is not evident in the absence of cryosurgery. PD-L1: programmed death-ligand 1.
FIG. 18 shows CPT&siR CRNPs without cryosurgery induce minimal expression of DAMPs in TME. Representative immunofluorescence images of HMGB1, CRT, HSP-70, and HSP-90 expression in left tumors harvested from mice following treatments with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the absence of cryosurgery. DAMP: damage-associated molecular patterns, TME: tumor microenvironment, HMGB1: high mobility group box protein 1, CRT: calreticulin, and HSP: heat shock protein. The experiments were repeated three times independently (n=3 mice) with similar results.
FIGS. 19A-19D show ICIE reverses the immunologically “cold” TME by decreasing the frequency of M2 and increasing the M1 frequency. FIGS. 19A-19B show representative flow cytometry plots of tumor associated macrophages (TAMs) M1 (F4/80+CD206−CD86+) and M2 (F4/80+CD206+CD86−) in left (FIG. 19A) or primary (FIG. 19B) tumors harvested from mice following treatments with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs without (a) or with (b) cryosurgery (n=3 mice). FIGS. 19C-19D, Percentage of M2 (FIG. 19C) and M1 (FIG. 19D) in left/primary tumors from mice injected with various formulations without/with cryosurgery (n=3 mice). Two-way ANOVA with Sidak's post-test and correction for multiple comparisons was used for statistical analyses. Data are presented as mean±SD (FIGS. 19C-19D).
FIGS. 20A-20D show ICIE reverses the immunologically “cold” TME by decreasing the frequency of M-MDSCs, PMN-MDSCs, and Treg cells in primary tumors. FIGS. 20A-B show representative flow cytometry plots of monocytic myeloid-derived suppressor cells (M-MDSC, CD11b+Ly6C+Ly6G−) in primary (FIG. 20A) or left (FIG. 20B) tumors harvested from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence (FIG. 20A) or absence (FIG. 20B) of cryosurgery (n=3 mice). FIGS. 20C-20D show representative flow cytometry plots of polymorphonuclear MDSCs (PMN-MDSC, CD11b+Ly6C−Ly6G+) in primary (FIG. 20C) or left (FIG. 20D) tumors harvested from mice injected with the various formulations mentioned above in the presence (c) or absence (d) of cryosurgery (n=3 mice). FIGS. 20E-20F, Representative flow cytometry plots of regulatory T (Treg) cells (CD4+Foxp3+) in primary (FIG. 20E) or left (FIG. 20F) tumors harvested from mice injected with the various formulations mentioned above in the presence (FIG. 20E) or absence (FIG. 20F) of cryosurgery (n=3 mice).
FIGS. 21A-21L show ICIE stimulates antitumor immune responses in primary tumors and activates the memory immune response against distant tumors. FIGS. 21A-21D show quantitative flow cytometry data of matured DCs (CD11c+CD86+) in inguinal lymph nodes (LNs, FIG. 21A), percentage of infiltrated CD8+ T cells (FIG. 21B), ratios of CD8+/Treg cells (FIG. 21C), and percentage of cytotoxic T cells (CTLs, CD8+GZMB+, FIG. 21D) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery (n=3 mice). FIG. 21E shows representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery (n=3 mice). FIG. 21F shows representative flow cytometry plots showing the percentage of effector memory T cell (TEM, CD3+CD8+CD44+CD62L−) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. FIG. 21G shows quantitative flow cytometry data of TEM in blood of mice with cryosurgery and injection of one of the different formulations (n=3 mice). FIG. 21H shows CD8+/Treg ratios in the blood of mice with cryosurgery and injection of one of the different formulations (n=3 mice). FIG. 21I shows representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. FIG. 21J shows quantitative flow cytometry data of CD8+/Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) of mice without cryosurgery (n=3 mice). All mice were injected with one of the indicated formulations. FIG. 21K shows quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery (n=3 mice). All mice were injected with one of the indicated formulations. FIG. 21L shows representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations (n=3 mice). Statistical analyses were done using two-way analysis of ANOVA with Sidak's post-test and correction for multiple comparisons. The experiments for FIG. 21E and 21I were repeated three times independently (n=3 mice) with similar results. Data are presented as mean±SD (FIGS. 21A-D, G-H, J-K).
FIGS. 22A-22G show ICIE induces strong antitumor immune response in primary tumors. FIGS. 22A-22B show representative flow cytometry plots of mature dendritic cells (DCs, CD11c+CD86+) in inguinal lymph nodes of mice injected with PBS, free CPT&siRs, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence (FIG. 22A) or absence (FIG. 22B) of cryosurgery (n=3 mice). FIGS. 22C-D show representative flow cytometry plots of CD4+ and CD8+ T cells in the primary (FIG. 22C) or left-side (FIG. 22D) tumors harvested from mice injected with various formulations in the presence (FIG. 22C) or absence (FIG. 22D) of cryosurgery (n=3 mice). FIGS. 22E-22F show representative flow cytometry plots of infiltrated cytotoxic T lymphocytes (CTLs, CD3+GZMB+) in the primary (FIG. 22E) or left-side (FIG. 22F) tumors harvested from mice injected with various formulations mentioned above in the presence (FIG. 22E) or absence (FIG. 22F) of cryosurgery (n=3 mice). FIG. 22G shows representative fluorescence staining of infiltrated CTLs (CD8+GZMB+) in the left-side tumors harvested from mice injected with various formulations in the absence of cryosurgery. The experiments were repeated three times independently (n=3 mice) with similar results. GZMB: granzyme B.
FIGS. 23A-23G show ICIE enhances the frequency of effector/central memory CD8+ T cells and increases the CD8+/Treg cell ratios in blood/spleen. FIG. 23A shows representative flow cytometry plots of effector memory T cells (TEM, CD3+CD8+CD44+CD62L−) in blood collected from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs and CPT&siR CRNPs in the absence of cryosurgery (n=3 mice). FIGS. 23B-23C, Representative flow cytometry plots of central memory T cells (TCM, CD3+CD8+CD44+CD62L+) in the spleen of mice injected with one of the aforementioned formulations in the presence (FIG. 23B) or absence (FIG. 23C) of cryosurgery. FIG. 23D shows quantitative data of TCM in the spleen of mice receiving treatment with one of the aforementioned formulations in the presence or absence of cryosurgery (n=3 mice). Statistical analyses were done using two-way ANOVA with Sidak's post-test and correction for multiple comparisons. Data are presented as mean±SD. FIGS. 23E-23F show representative flow cytometry plots of CD4+ and CD8+ T cells in blood harvested from mice injected with one of the various formulations in the presence (FIG. 23E) or absence (FIG. 23F) of cryosurgery (n=3 mice). FIG. 23G shows representative flow cytometry plots of Treg cells (CD4+Foxp3+) in the blood collected from mice injected with the various formulations in the absence of cryosurgery (n=3 mice).
FIGS. 24A-24G show ICIE stimulates strong antitumor immune response in distant tumors. FIGS. 24A-24B shows representative flow cytometry plots of Treg cells (CD4+Foxp3+) in distant (with cryosurgery on primary tumor)/right-side (without cryosurgery) tumors harvested from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs and CPT&siR CRNPs in the presence (FIG. 24A) or absence (FIG. 24B) of cryosurgery (on primary tumor) (n=3 mice). FIGS. 24C-24D show representative flow cytometry plots of CD4+ and CD3+ T cells in distant/right-side tumors harvested from mice injected with various formulations in the presence (FIG. 24C) or absence (FIG. 24D) of cryosurgery (on primary tumor) (n=3 mice). FIGS. 24E-24F, Representative flow cytometry plots of infiltrated CTLs (CD8+GZMB+) in the distant/right-side tumors harvested from mice injected with various formulations in the presence (FIG. 24E) or absence (FIG. 24F) of cryosurgery (on primary tumor) (n=3 mice). FIG. 24G shows representative immunofluorescence staining of infiltrated CTL (CD8+GZMB+) in the right tumors harvested from mice injected with various formulations in the absence of cryosurgery. The experiments were repeated three times independently (n=3 mice) with similar results.
FIGS. 25A-25E show ICIE effectively inhibits tumor growth in both primary and distant tumors with no evident systemic toxicity. FIG. 25A shows tumor growth curve of left-side tumors including primary (P) tumors from mice with cryosurgery and left (L) tumors from mice without cryosurgery (n=6 mice). The mice were also injected with one of the formulations including: PBS, free CPT&siR, CPT CRNPs, siR CRNPs, or CPT&siR CRNPs. FIG. 25B shows tumor growth curve of right-side tumors including distant (D) tumors from mice with cryosurgery on P tumors and right (R) tumors from mice without cryosurgery (n=6 mice). The mice were also injected with one of the indicated formulations. FIGS. 25C-25D show weight of left-side (L versus P, FIG. 25C) and right-side (R versus D, FIG. 25D) tumors (n=6 mice) obtained after sacrificing the mice at the end of the study, showing effective tumor destruction by the ICIE treatment. FIG. 25E shows representative hematoxylin and eosin (H&E) staining images of left-side (L versus P) and right-side (R versus D) tumors, showing extensive necrosis in both the primary and distant tumors from mice with the ICIE treatment. The experiments were repeated three times independently (n=3 mice) with similar results. Statistical analyses were done using two-way analysis of ANOVA with Sidak's post-test and correction for multiple comparisons. Data are presented as mean±SD (FIGS. 25C-25D).
FIGS. 26A-26D show ICIE inhibits the growth of both primary and distant tumors. FIGS. 26A-26B show primary (with cryosurgery, a)/left-side (without cryosurgery, b) tumor growth curves in mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence (FIG. 26A) or absence (FIG. 26B) of cryosurgery (n=6 mice). FIGS. 26C-26D show distant (with cryosurgery on primary tumor, FIG. 26C)/right-side (without cryosurgery, FIG. 26D) tumor growth curves in mice injected with various formulations in the presence or absence of cryosurgery (on primary tumor) (n=6 mice). Two-way ANOVA with Sidak's post-test and correction for multiple comparisons was used for statistical analyses. Data are presented as mean±SD (FIGS. 26A-26D).
FIGS. 27A-27C show Images of tumors showing ICIE reduces the size of both primary and distant tumors. FIG. 27A shows representative photographs of EO771 tumor-bearing mice at the end of the study, showing effective tumor destruction by the ICIE treatment. The black regions are a result of skin wound that often occurs in the nipple area due to orthotopic tumor growth. The experiments were repeated six times independently (n=6 mice) with similar results. FIG. 27B shows images of primary and distant tumors collected on the final day of the study. Mice in different groups were treated with cryosurgery and injections of PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs (n=6 mice). FIG. 27C shows images of left and right tumors collected on the final day of the study. Mice were injected with the various formulations without cryosurgery (n=6 mice).
FIGS. 28A-28B show ICIE significantly decreases the expression of Ki-67 and CD31 in tumors. FIG. 28A shows representative immunofluorescence staining of Ki-67 in the primary (P, with cryosurgery)/left-side (L, without cryosurgery) tumor sections collected from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence or absence of cryosurgery. FIG. 28B shows representative immunofluorescence staining of CD31 in the primary (P, with cryosurgery)/left-side (L, without cryosurgery) tumor sections collected from mice injected with various formulations in the presence or absence of cryosurgery. All the experiments were repeated three times independently (n=3 mice) with similar results.
FIGS. 29A-29B show ICIE induces no significant changes in body weight. FIG. 29A shows body weight recorded for mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence of cryosurgery (n=6 mice). FIG. 29B shows body weight recorded for mice injected with various formulations in the absence of cryosurgery (n=6 mice). Two-way ANOVA with Sidak's post-test and correction for multiple comparisons was used for statistical analyses. ns: not significance. Data are presented as mean±SD (FIGS. 29A-29B).
FIG. 30 shows ICIE induces no evident damage in major organs. H&E staining of major organs including heart, liver, spleen, lung, and kidney collected from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the presence of cryosurgery. H&E: hematoxylin and eosin. The experiments were repeated three times independently (n=3 mice) with similar results.
FIG. 31 shows CRNPs induce no evident damages in major organs. H&E staining of major organs including heart, liver, spleen, lung, and kidney collected from mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs in the absence of cryosurgery. The experiments were repeated three times independently (n=3 mice) with similar results.
FIGS. 32A-32B show CRNPs induce no evident changes in blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. FIGS. 32A-32B show detection of ALT (FIG. 32A) and AST (FIG. 32B) levels in the blood of mice injected with PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs at the end of the study (n=4 mice). Statistical analyses were performed using one-way ANOVA with Tukey's multiple comparisons and correction. ns: not significant. Data are presented as mean±SD (FIGS. 32A-32B).
FIGS. 33A-33F show ICIE generates long-term antitumor memory immune response and inhibits metastasis in the lung. FIG. 33A shows a schematic illustration of the in vivo experimental design. Mice received intravenous injection of various formulations every three (3) days. Cryosurgery was performed on the orthotopic tumor (OT) at eight hours (8 h) after the first injection of the various formulations. Lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day ten (10) after the first injection of the different formulations. FIG. 33B shows weight of lungs collected from mice at the end of the study, showing prevention of lung metastasis by ICIE (n=6 mice). FIG. 33C shows representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. FIG. 33D shows quantification of lung metastasis nodes from mice at the end of the study (n=6 mice). FIG. 33E shows representative H&E staining images of lung tissues collected at the end of the study, showing decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 μm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently (n=3 mice) with similar results. FIG. 33F shows the percentage of effector memory T cells (TEM, CD3+CD8+CD44+CD62L−) in blood collected from mice with injection of one of the various formulations (n=3 mice). Statistical analyses were performed using one-way analysis of ANOVA with Tukey's multiple comparisons test and correction. Data are presented as mean±SD (FIG. 33B, 33D, 33F).
FIGS. 34A-34B show ICIE effectively inhibits the growth of orthotopic tumors (OTs) and generates long-term antitumor memory immune response against metastatic tumors. FIG. 34A shows weight of OTs obtained after sacrificing the mice at the end of the study, showing effective destruction of localized OTs by the ICIE treatment (n=6 mice). Mice with OTs were treated with PBS, PBS+cryo, CPT&siR CRNPs, or CPT&siR CRNPs+cryo. Cryosurgery (cryo) was performed on the OT at eight hours (8 h) after the first injection of the various formulations. FIGS. 34B shows representative flow cytometry plots of effector memory T cells (TEM, CD3+CD8+CD44+CD62L−) in blood collected from mice with no OT injected with PBS, and mice with OT treated with PBS, PBS+cryo, CPT&siR CRNPs, or CPT&siR CRNPs+cryo. Cryosurgery was performed on the OT at eight hours (8 h) after the first injection of the various formulations (n=3 mice). Statistical analyses were performed using one-way ANOVA with Tukey's multiple comparisons and correction. Data are presented as mean±SD (FIG. 34A).
FIG. 35 shows ICIE effectively prolongs the overall survival of Balb/c mice with metastatic tumors. Overall survival rate of Balb/c mice with 4T1 lung metastasis in mice with no orthotopic tumor (OT) after treatment with PBS, and mice with OT after treating with PBS, PBS+cryo, CPT&siR CRNPs, or CPT&siR CRNPs+cryo (n=8 mice). Cryosurgery was performed on the OT at eight hours (8 h) after the first injection of the various formulations. Statistical analyses were performed using the Log-rank (Mantel-Cox) test.
An artisan of ordinary skill in the art need not view, within isolated figure(s), the near infinite distinct combinations of features described in the following detailed description to facilitate an understanding of the present disclosure.
DETAILED DESCRIPTION
The present disclosure is not to be limited to that described herein. Mechanical, electrical, chemical, procedural, and/or other changes can be made without departing from the spirit and scope of the present disclosure. No features shown or described are essential to permit basic operation of the present disclosure unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used above have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present disclosure pertain.
The terms “a,” “an,” and “the” include both singular and plural referents.
The term “or” is synonymous with “and/or” and means any one member or combination of members of a particular list.
As used herein, the term “exemplary” refers to an example, an instance, or an illustration, and does not indicate a most preferred embodiment unless otherwise stated.
The term “about” as used herein refers to slight variations in numerical quantities with respect to any quantifiable variable. Inadvertent error can occur, for example, through use of typical measuring techniques or equipment or from differences in the manufacture, source, or purity of components.
The term “substantially” refers to a great or significant extent. “Substantially” can thus refer to a plurality, majority, and/or a supermajority of said quantifiable variables, given proper context.
The term “generally” encompasses both “about” and “substantially.”
The term “configured” describes structure capable of performing a task or adopting a particular configuration. The term “configured” can be used interchangeably with other similar phrases, such as constructed, arranged, adapted, manufactured, and the like.
Terms characterizing sequential order, a position, and/or an orientation are not limiting and are only referenced according to the views presented.
Synthesis and Characterization of CRNPs for Cold-Triggered Release
Previous studies used polymers with a lower critical solution temperature (LCST) of ˜14-30° C. to synthesize thermally responsive nanoparticles, which can't be used to precisely control the drug release within the frozen tumor iceball while sparing the surrounding normal tissue in response to the cold temperature (˜negative four degrees Celsius (−4° C.)) at the outer surface of an iceball during cryosurgery. Therefore, a series of poly (N-isopropylacrylamide copolymerized with butyl acrylate) (pNIPAAm-BA) polymers were synthesized with varying LCSTs by adjusting the feeding ratio of NIPAAm to BA. Proton nuclear magnetic resonance (1H-NMR) spectroscopy analyses of chemical bonds show successful synthesis of the pNIPAAm-BA polymers (FIG. 1A). The number-averaged molecular weight (Mn) ranges from ˜67 to 83 kDa as determined by gel permeation chromatography (GPC, FIGS. 1B-1C). The LCST of the polymers dispersed in different solutions was measured with cryo-microscopy. As shown in FIG. 1D, the LCST of the same polymer displays no significant variation in different solvents/solutions, but a higher NIPAAm content leads to a higher LCST of the pNIPAAm-BA polymer. The resultant p(NIPAAm)589-co-(BA)117, with an LCST of −4.4±0.6° C. that is close to the temperature on the outer surface of a frozen tumor iceball, was selected to synthesize the CRNPs for co-encapsulating irinotecan (CPT, a clinically used chemotherapy drug) and programmed death-ligand 1 (PD-L1) silencing siRNA (siR) using a double-emulsion method (FIG. 2). Other materials used for synthesizing the CRNPs include Poly (D, L-lactide-co-glycolide) (PLGA), Pluronic F127 (PF127), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and chitosan. Most of the materials are either FDA-approved for medical use (PLGA, PF127, DPPC) or naturally derived material (chitosan) with good biocompatibility, which may facilitate the clinical translation of the CRNPs. The surface of the resultant CPT&siR-laden CRNPs (or CPT&siR CRNPs for short) is decorated with chitosan (CS) for enhanced tumor-targeting capability.
To optimize the loading of CPT and siR in the CRNPs, the encapsulation efficiency (EE) and loading content (LC) of CPT were measured for feeding CPT at 1, 3, and 5% of the polymers. As shown in FIGS. 3A-3B, the EE of CPT decreases monotonically with the increase of the CPT feeding percentage, while the LC of CPT reaches a plateau at 3% feeding percentage. Therefore, the 3% CPT feeding percentage was used in this work, for which the LC of CPT is 1.5±0.2% with an encapsulation efficiency (EE) of 51.2±8.2%. For siR, the EE and LC are 73.4±4.2% and 0.14±0.01%, respectively. The particle size of the optimized CPT&siR CRNPs is 143.6±6.6 nm in diameter with a surface zeta potential of −4.9±0.4 mV. Transmission electron microscopy (TEM) analysis of the CPT&siR CRNPs shows that they have a core-shell structure (at twenty two degrees Celsius (22° C.) with no cooling, FIG. 4A). Furthermore, they are stable over time and show negligible changes in their size after being suspended in phosphate buffered saline (PBS) for five days (FIG. 3C). Moreover, compared to the free siR that easily gets degraded within five minutes (5 min) of incubation with PBS at twenty two degrees Celsius (22° C.) (FIG. 3D), the siR encapsulated in the CPT&siR CRNPs remains stable for at least twenty four hours (24 h) (FIG. 3E).
The cold-responsiveness of the CPT&siR CRNPs was studied by TEM and scanning electron microscopy (SEM) first. As shown in the TEM and SEM images in FIG. 4A, destruction of the structure of the CRNPs after cold treatment (incubating the samples at negative four degrees Celsius (−4° C.) for ten minutes (10 min) and warming back to twenty two degrees Celsius (22° C.)) is evident. The cold-induced destruction of the CRNP structure triggered the release of the encapsulated siR, resulting in an evident band (white) of siR in agarose gel for the CPT&siR CRNPs with the cold treatment at 4° C. (FIG. 5A). The hydrodynamic diameter of the CPT&siR CRNPs is greatly increased with two wide peaks (one at ˜700 nm and the other at ˜5000 nm on average) after the cold treatment (FIG. 4B), probably due to aggregation of the hydrophobic polymers/drugs dissembled from the CPT&siR CRNPs. According to the photographs and cryo-microscopy images of the nanoparticle samples in deionized water (DW, 12.0 mg ml−1, FIG. 4C), the aqueous sample of CPT&siR-laden Poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (CPT&siR PLGA NPs, 135.7±6.1 nm in diameter with a surface zeta potential of −14.1±0.6 mV) that are not cold-responsive, has a homogeneous and milky appearance and blocks light to give a dark cryo-microscopic image at both twenty two degrees Celsius (22° C.) (either before or after cooling) and negative four degrees Celsius (−4° C.). In contrast, a homogeneous and transparent aqueous sample can be observed for the CPT&siR CRNPs at negative four degrees Celsius (−4° C.), although the sample is also homogeneous and milky at twenty two degrees Celsius (22° C.) before the cooling treatment. The cryo-microscopic images showing the cold-triggered changes were further analyzed to quantify their grayscale intensity. When slowly cooled (1° C. min−1) below ˜4° C., the sample of the CPT&siR CRNPs becomes more and more transparent with lower and lower grayscale intensity (FIG. 4D). In comparison, the grayscale intensity of the microscopic images for the sample of the CPT&siR PLGA NPs remained constant during cooling. When warmed back to twenty two degrees Celsius (22° C.), the sample with CPT&siR CRNPs becomes heterogeneous with a large whitish aggregate of insoluble materials piling up at the bottom (FIG. 4C). These results confirm the cold-triggered dissolution and irreversible disassembly of the CPT&siR CRNPs.
To determine the cold-triggered drug release profile, the CPT&siR CRNPs were incubated in different buffers at pH 5.0, 6.5, and 7.4 to mimic that in lysosomes, the extracellular space of tumor, and normal tissue, respectively. Before applying the cold treatment, both CPT and siR are released in a sustained manner at thirty seven degrees Celsius (37° C.), with less than 10% release in eight hours (8 h) (FIGS. 5B-5D). After cold treatment by incubating the samples at negative four degrees Celsius (−4° C.) for ten minutes (10 min), cold-triggered rapid release profiles are observed for both CPT and siR in all the three different buffers, due to the cold-induced disassembly of the CRNPs (FIG. 4A, 4C).
Cold-Triggered Endo/Lysosomal Escape of siR and Enhancement of PD-L1 Silencing
To investigate cellular uptake and intracellular trafficking of the CRNPs, CRNPs loaded with CPT and cyanine 5 (Cy5)-labeled siR were incubated with green fluorescent protein positive EO771 (GFP+ EO771) breast cancer cells and lymphocytes from mouse spleen at thirty seven degrees Celsius (37° C.) for eight hours (8 h). Significant red fluorescence signal of Cy5 can be observed in the GFP+ EO771 cells with confocal microscopy, indicating successful uptake of the CPT&Cy5-siR CRNPs by the cells (FIG. 6A). In contrast, negligible Cy5 fluorescence could be detected in the cells incubated with free CPT and Cy5-siR, probably due to degradation and/or poor uptake of the siR. Successful cellular uptake of the CPT&Cy5-siR CRNPs is confirmed by flow cytometry, which shows over 90% of the GFP+ EO771 cells are Cy5 positive (FIGS. 6B-6C). For the non-cancerous cells like lymphocytes isolated from the spleen, only ˜0.9% of them are Cy5 positive (FIG. 6D), suggesting the good cancer cell targeting capability of the CRNPs. Cold treatment of CRNPs in cells can trigger endo/lysosomal escape of the siR into the cytosol. Without cold treatment (CPT&Cy5-siR CRNPs), Cy5 fluorescence can be observed in the endo/lysosomes of EO771 cells after eight hours (8 h) of incubation (FIG. 4E), indicated by the evident overlap of Cy5 (red) and endo/lysosomes (green) as shown by the confocal images, the distribution of the red and green fluorescence intensities, and the high intensity of Cy5-siR fluorescence in the endo/lysosomal areas. After cold treatment (CPT&Cy5-siR CRNPs+C), colocation of Cy5 and endo/lysosomes is decreased with an evident separation of red and green fluorescence signals, indicating successful cold-triggered escape of Cy5-siR from endo/lysosomes into the cytosol. The cold-triggered enhancement of endo/lysosomal escape is due to the encapsulation of sodium chloride (NaCl) in the CRNPs. In EO771 cells treated with the CRNPs containing no NaCl, cold treatment (CPT&Cy5-siR CRNPs+C, no NaCl) does not induce an evident separation between the red fluorescence of Cy5-siR and the green fluorescence of endo/lysosomes (FIG. 7). The underlying mechanism for NaCl-induced endo/lysosomal escape may be ascribed to the reason that the cold treatment triggers rapid release of the encapsulated Na+ and Cl− into the endo/lysosomes, resulting in a surge of osmolality (hypertonicity) and influx of water to destabilize or rupture endo/lysosomes. As a result, the encapsulated CPT and PD-L1 silencing siRNA are rapidly released into the cytosol to perform the chemotherapy and gene silencing functions.
To investigate the gene silencing effect of CRNPs-delivered siR, we incubated GFP+ EO771 cells with CRNPs containing GFP silencing siRNA (GFP-siR CRNPs) for eight hours (8 h), cold-treated the cells at negative four degrees Celsius (−4° C.) for ten minutes (10 min), and further cultured them for 40 h. The cold-triggered GFP-siR release from the CRNPs markedly decreases the expression of GFP in the GFP EO771 cells, according to the data of confocal microscopy (FIG. 8) and flow cytometry (FIG. 4F-4G). Without cold treatment, GFP fluorescence is eliminated in ˜26% of GFP+ EO771 cells treated with the GFP-siR CRNPs. After cold treatment of the cells incubated with GFP-siR (CRNPs+C), the percent of cells with silenced GFP expression increases to ˜95%, indicating cold-triggered siR release and its effective silencing of GFP expression in the cells. This is further confirmed by treating EO771 cells with CRNPs loaded with siR specific for silencing PD-L1. Cold-triggered release of the PD-L1 siR from CRNPs (PD-L1-siR CRNPs+C) results in a much lower expression of PD-L1 in EO771 cells (2.5%) than the treatments of free PD-L1 siR and PD-L1-siR CRNPs without cooling (FIG. 4H-4I). These results indicate that the cold-triggered siR release from CRNPs significantly enhances their gene silencing ability.
To evaluate the anticancer capacity of CPT&siR CRNPs, EO771 cells were incubated with blank CRNPs, free CPT&siR, and CPT&siR CRNPs at various CPT concentrations for twenty four hours (24 h) with and without cold treatment. CPT&siR CRNPs show significantly higher toxicity to the cancer cells than free CPT&siR (FIG. 9), probably due to poor cellular uptake of free CPT&siR. Importantly, cold treatment further significantly enhances the toxicity of CPT&siR CRNPs to cancer cell, showing the cold-triggered rapid release of CPT is more effective in killing cancer cells than the slow and sustained release of CPT from the nanoparticles. It is worth noting that the viability of cells treated with the blank CRNPs (up to 5.6 mg ml−1) is higher than ˜90%. The cancer cell killing efficacy of cold-treated CPT&siR
CRNPs at a CPT concentration of 10.0 μg ml is further confirmed by flow cytometry data, showing a high percentage of apoptosis/necrosis (77.9%) in EO771 cells with the CPT&siR CRNPs+C treatment (FIG. 10).
ICIE Increases the Expression of DAMPs and Maturation of DCs
We next studied the impact of ICIE (i.e., the CPT&siR CRNPs+C treatment) on ICD via examining the expression of DAMPs and maturation of DCs. For cold treatment, EO771 cells were incubated with the CPT&siR CRNPs or other control formulations including PBS, free CPT&siR, CPT-laden CRNPs (CPT CRNPs), and PD-L1 siR-laden CRNPs (siR CRNPs) for eight hours (8 h), cold-treated the cells at negative four degrees Celsius (−4° C.) or negative twenty degrees Celsius (−20° C.) for ten minutes (10 min), and further cultured them for 16 h. Flow cytometry was used to quantify the expression of DAMPs in cells, including heat shock protein-90 (HSP-90), heat shock protein-70 (HSP-70), calreticulin (CRT), and high-mobility group box 1 protein (HMGB1), following the different treatments. Cold treatments (at both negative four degrees Celsius (−4° C.) and negative twenty degrees Celsius (−20° C.)) significantly enhance production of all the DAMPs for all the formulations, compared to thirty seven degrees Celsius (37° C.) (FIG. 11A). These data indicate that cold treatment alone can promote DAMP expression and induce ICD. Particularly, the formulation of CPT&siR CRNPs with cold treatments (i.e., ICIE) at negative four degrees Celsius (−4° C.) and negative twenty degrees Celsius (−20° C.) induce higher expression of HSP-70, HSP-90, HMGB1, and CRT than all the other formulations with the same cold treatment, indicating that cold-triggered release of both CPT and siR from the CPT&siR CRNPs helps boost ICD beyond the cold treatment alone. When the frozen tumor cells undergo ICD, the released DAMPs promote recruitment, accumulation, as well as maturation of DCs in the TME, which should enhance the engulfment of tumor antigens by DCs to facilitate antigen presentation to T cells. This eventually should result in activation of CD8+ CTLs to kill tumor cells, as schematically illustrated in FIG. 11B.
The activation of an antitumor immune response requires the maturation of DCs and presentation of tumor antigens to T cells. To this end, maturation of DCs and the capability of further activating CD8+ T cells (that can recognize the OVA antigen) were evaluated by co-culture of DCs and cancer cells (EO771-OVA) with various treatments. As shown in FIGS. 11C-11D, ICIE (CPT&siR CRNPs+C) treatment at negative twenty degrees Celsius (−20° C.) elevates the maturation of bone marrow dendritic cells (BMDCs, CD11c+CD86+) to 49.4%. This level is significantly higher than that for the CPT&siR CRNPs (no cold treatment, by default), free CPT&siR (no cold treatment, by default), and PBS (no cold treatment, by default). Antigen presentation capabilities in these mature BMDCs were examined by detecting the level of ovalbumin-derived peptide with the amino sequence of SIINFEKL that binds to the H-2Kb of major histocompatibility complex I (MHC I) molecule (to result in Kb-SIINFEKL) in antigen presenting cells (APCs). BMDCs co-cultured with the EO771-OVA cells treated with ICIE express the highest amount of SIINFEKL (58.9%, FIGS. 11E-11F). Indeed, this is significantly higher than that for BMDCs co-cultured with the EO771-OVA cells treated with CPT&siR CRNPs, free CPT&siR, and PBS (31.2, 17.0, and 3.1%, respectively, FIGS. 11E-11F). Moreover, BMDCs co-cultured with the EO771-OVA cells received ICIE treatment secrete 2.3-, and 1.6-fold more pro-inflammatory cytokines (IFN-γ and TNF-α) than BMDCs co-cultured with the EO771-OVA cells received CPT&siR CRNPs without cold treatment (FIGS. 12A-12B), suggesting the potentiation of antitumor immune response when combining cryosurgery with CPT&siR CRNPs.
ICIE Enhances the Proliferation, Activation, and Tumor-Attacking Efficacy of CD8+ T Cells
When co-cultured with CD8+ T cells, BMDCs matured by EO771-OVA cells treated with ICIE at negative twenty degrees Celsius (−20° C.) substantially increase the proliferation of CD8+ T cells (78.9%, FIGS. 11G-11H), which is significant compared to the treatments of CPT&siR CRNPs, free CPT&siR, and PBS (34.9, 16.4, and 8.3%, respectively). To investigate the activation of CD8+ T cells, we examined the expression of cytotoxic granzyme B (GZMB, a serine protease commonly found in the granules of CTLs) using flow cytometry. Indeed, ICIE increases the percentage of CD8+GZMB+to 56.5%, which is significantly higher than that for CPT&siR CRNPs, free CPT&siR, and PBS (36.1, 26.4, and 17.0%, respectively, FIGS. 11I-11J).
We next tested the tumor-attacking capability of the activated CD8+ T cells. CD8+ T cells activated by BMDCs co-cultured with EO771-OVA cells receiving ICIE treatment, migrate to and accumulate around the EO771-OVA tumor cells (labeled with green color) within five minutes (5 min) to attack them persistently and the tumor cells are eventually dismantled within 4 h (FIGS. 11K). Furthermore, CD8+ T cells from the ICIE group result in a significantly higher percentage of cancer cell death (79.9%) than the cells from the CPT&siR CRNPs (30.0%) group (FIGS. 11L-11M). CD8+ T cells from the PBS and free CPT&siR groups cause 16.1% and 17.7% of EO771-OVA cell death, respectively. To assess the synergistic effects of co-delivery of CPT and siR, we compared the cancer cell killing capability of CPT&siR CRNPs (either with or without cold treatment) with that CPT CRNPs alone, siR CRNPs alone, and the simple addition (i.e., additive effect) of the treatments with CPT CRNPs alone and siR CRNPs alone. In the absence of cold treatment, co-delivery of the two agents in the CPT&siR CRNPs show no synergistic effect (FIGS. 13A-13B): It leads to ˜31% of cancer cell death, which is less than the percentage of cancer cell death (˜39%) due to the additive effect of the CPT CRNPs alone and siR CRNPs alone. Importantly, when cold treatment is applied, the synergistic effect of CPT and siR in CPT&siR CRNPs is evident (FIGS. 13C-13D): It causes a significantly higher percentage of cancer cell death (80.5%) than that (61.4%) due to the additive anticancer effect of CPT CRNPs alone and siR CRNPs alone. These in vitro results collectively show that ICIE holds great potential for activating T cells and enhancing their tumor killing functionality.
CRNPs Accumulate in Tumor and Exhibit Negligible Hemolysis
To explore the in vivo distribution of CRNPs, an orthotopic mouse breast tumor model was created by injecting 1×106 EO771 cells per mouse into the left abdominal mammary fat pad of C57BL/6 mice to grow tumors (one per mouse) into approximately 100 mm3 (at around ten (10) days). After intravenous injection of various formulations (CPT&Cy5-siR CRNPs, free CPT&Cy5-siR, and PBS) into the mice bearing EO771 tumors, Cy5 fluorescence in the mice was studied by whole animal imaging. As shown in FIGS. 14A-14B, Cy5 fluorescence is evidently observable in the tumor areas (indicated by blue arrows) at four hours (4 h), eight hours (8 h), twelve hours (12 h), and twenty four hours (24 h) only for the group of CPT&Cy5-siR CRNPs. At each time point, blood was drawn from the mice for testing Cy5 fluorescence in circulation. Little Cy5 fluorescence signal is observable/detectable (decreased by one order of magnitude) after 8-12 h for the free CPT&Cy5-siR group (FIG. 14C, FIG. 15). In contrast, for the CPT&Cy5-siR CRNPs group, the Cy5 fluorescence is evident even at twenty four hours (24 h), showing encapsulation of the Cy5-siR in the CRNPs can prolong its time in blood circulation because the nanoparticle can protect the siR from degradation (FIG. 3E). The fluorescence of Cy5 in different organs was further examined after sacrificing the mice at twenty four hours (24 h). Mice treated with free CPT&Cy5-siR show minimal Cy5 fluorescence in tumors, but high Cy5 fluorescence in kidneys and liver and moderate-low fluorescence in lung and spleen (FIGS. 14D-14E). In contrast, the Cy5 fluorescence is significantly reduced in kidneys, and significantly increased in liver for the CPT&Cy5-siR CRNPs group (which is not unusual for nanoparticles for drug delivery), compared to the free CPT&Cy5-siR group. Most importantly, the Cy5 fluorescence in tumors is significantly higher for the CPT&Cy5-siR CRNPs group than the free CPT&Cy5-siR group, indicating the tumor accumulating/targeting capability of the CRNPs. Although CPT&Cy5-siR CRNPs shows relatively higher liver accumulation than free CPT&Cy5-siR, drug release from the CRNPs in liver where no cryosurgery will be applied, would be minimal to minimize any potential toxicity of the two encapsulated agents.
Since the CRNPs need to go through long blood circulation after intravenous (i.v.) injection, their blood biocompatibility was further examined with the hemolysis test. As shown in FIG. 14F, CPT&siR CRNPs of 25-800 μg ml−1 (in PBS) induce a negligible percentage of hemolysis comparable to PBS while DW causes nearly complete hemolysis. Furthermore, compared to PBS, CPT&siR CRNPs (800 μg ml−1) do not cause any morphological changes to the red blood cells (SEM images, FIG. 14F) after incubation with the whole blood at thirty seven degrees Celsius (37° C.) for twenty four hours (24 h). These data show that no evident blood incompatibility is observable for the CRNPs.
ICIE Turns the TME from Immunologically “Cold” into “Hot” in Primary Tumors
To create an orthotopic breast tumor model, C57BLC/6 mice were inoculated with EO771 cells (1×106 cells per gland) in the fat pad of both the left and right abdominal mammary glands. After ten (10) days of tumor establishment, various formulations (PBS, free CPT&siR, CPT CRNPs, siR CRNPs, and CPT&siR CRNPs) were injected via the tail vein every three days for a total of nine injections either without (FIG. 16A) or with (FIG. 16B) cryosurgery. For the groups with cryosurgery, it was done on the left (in terms of mice) tumors once (blue arrow, FIG. 16B) at eight hours (8 h) after the first injection of the formulations by gently pressing the cryoprobe against the tumor at its central location (FIG. 16C). Therefore, the left tumors are called primary ones with cryosurgery while the right tumors without cryosurgery are called distant ones for all groups with cryosurgery. It is worth noting that the treatment with cryosurgery being conducted only once and the formulations being injected multiple times, is similar to how frequent cryosurgery (usually once) and chemotherapy (almost always multiple times) are conducted in the clinic. To monitor the temperature during the ten minutes (10 min) of cryosurgery, we attached two K-type thermocouples (TCs) on the surface of the primary tumor (FIG. 16C): one for measuring the temperature at the tumor boundary (TC1) and the other (TC2) for monitoring the temperature at the central location of the tumor next to the cryoprobe. During cryosurgery, the temperature at tumor boundary and central locations was kept at approximately negative four degrees Celsius (−4° C.) and negative twenty degrees Celsius (−20° C.) for ten minutes (10 min), respectively (FIG. 16D). This was achieved by turning the cryoprobe on and off intermittently to maintain the iceball roughly within the tumor boundary during the cryosurgery procedure. Moreover, a FLIR infrared camera was used to monitor the temperature in the entire primary tumor area (indicated by dashed circles, FIG. 16E) during cryosurgery. Typical temperature contours in the tumor area together with the temperature on the tumor boundary at 0 s (before cryosurgery), 300 s (during cryosurgery), and 630 s (after cryosurgery and warming up) are also given in FIG. 16E. Typical image showing frozen tumor iceball formation is also captured at 300 s during cryosurgery (for which the cryoprobe and TCs are temporarily removed).
After the aforementioned treatments, their impact on the TME in the primary tumors was studied. First, the PD-L1 silencing effect of siR in the primary (for mice with cryosurgery) and left (for mice without cryosurgery) tumors was determined by western blot. Both ICIE (i.e., CPT&siR CRNPs with cryosurgery) and siR CRNPs with cryosurgery treatments significantly decrease the PD-L1 expression in tumors when compared with CPT CRNPs with cryosurgery or PBS with cryosurgery (FIG. 17A). In the absence of cryosurgery, the silencing effect is not evident for all the formulations including siR CRNPs or CPT&siR CRNPs (FIG. 17B). Second, the expression of ICD markers (i.e., DAMPs including HMGB1, CRT, HSP-70, and HSP-90) is markedly increased in the primary tumors from the groups with cryosurgery (FIG. 16F), compared to their counterparts without cryosurgery (FIG. 18). Notably, The ICIE treatment stimulates higher expression of all the ICD markers in the primary tumors than all the other treatments with or without cryosurgery. Third, compared to the percentage of tumor associated macrophages (TAMs) in left tumors (corresponding to primary tumors in mice with cryosurgery) of mice without cryosurgery, cryosurgery not only decreases the population of pro-tumorigenic M2 TAMs (F4/80+CD206+CD86−) but also elevates the percentage of anti-tumorigenic M1 TAMs (F4/80+CD206−CD86+) in all treated groups (FIGS. 19A-19D). As a result, a marked increase in the M1/M2 ratios is observable in mice with cryosurgery (FIG. 16G), with the highest M1/M2 ratio of 4.0±0.5 in mice receiving the ICIE treatment. Fourth, cryosurgery evidently attenuates the frequency of both monocytic myeloid-derived suppressor cells (M-MDSCs, CD11+Ly6C+Ly6G−) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b+Ly6C−Ly6G+) that perform their immunosuppressive activities within the TME (FIGS. 16H-16I and FIGS. 20A-20D). The ICIE treatment decreases the percentage of M-MDSCs and PMN-MDSCs to 8.8±1.0% and 16.1±2.1%, respectively, which is significantly lower than that for its counterpart (CPT&siR CRNPs) with no cryosurgery (18.9±1.4% for M-MDSCs and 35.4±3.5% for PMN-MDSCs). Furthermore, in the primary tumor, the percentage of regulatory T (Treg, CD4+Foxp3+) cells is greatly reduced by the ICIE treatment (FIG. 16J and FIGS. 20E-20F). The PBS control group (without cryosurgery) had a high percentage of Treg (42.9±3.0%), indicating an immunosuppressive TME. The cryosurgery treatment can significantly reduce the percentage of Treg cells for all the formulations. Importantly, the ICIE treatment results in the most reduction of the Treg cell percentage to only 1.6±0.7% (FIG. 16J and FIG. 20E). Collectively, these data support that the ICIE treatment induces ICD; produces tumor antigens; and decreases the frequency of immunosuppressive M2, MDSCs, and Tregs; to reverse the immunologically cold TME. This is attributed to the cryosurgery-induced ICD reinforced by the cold-triggered rapid release of both the chemotherapy drug irinotecan (CPT) that inhibits DNA synthesis, and the siRNA (siR) that silences the overexpression of T cell inhibitory ligand PD-L1 on cancer cells to remove the immune checkpoint that inhibits the activation of CD8+ T cells. In addition, the frostbite effect of cryosurgery might kill the M2, MDSCs, and Tregs in the immunosuppressive TME, to reduce their frequency in the tumor after cryosurgery. As a result, ICIE is highly effective to turn the immunologically “cold” TME into a hot one.
ICIE Boosts the Antitumor Immune Responses in Primary Tumors
To assess if the aforementioned modulation of TME by ICIE boosts the antitumor immune responses, we analyzed the population of DCs and CD8+ T cells in the mice. As shown in FIG. 21A and FIGS. 22A-22B, cryosurgery promotes maturation of DCs in the inguinal lymph nodes, leading to significantly increased percentages of mature DCs (CD11c+CD86+) for all the groups with cryosurgery. In particular, the ICIE treatment (i.e., CPT&siR CRNPs with cryosurgery) triggers the highest percentage of DC maturation among all the treatments (with or without cryosurgery). Furthermore, in the primary tumors where the cryosurgery is applied, the augmented DC maturation enables significantly higher accumulation of tumor-infiltrating lymphocytes (FIG. 21B and FIG. 22C). Importantly, the percentage of tumor-infiltrating CD8 T cells for the ICIE group is the highest (23.8±0.6% out of all T cells) among all groups, with a highest CD8/Treg ratio of 17.6±10.3 (FIG. 22C) that is 117 times of the CD8+/Treg ratio in the L tumor with the PBS treatment alone. This indicates the ICIE treatment turns the immunologically “cold” TME into a “hot” one. In contrast, the treatment of CPT&siR CRNPs without cryosurgery only results in 9.5±0.8% of tumor infiltrating CD8+ T cells (FIG. 22D) and a CD8+/Treg ratio of 0.4 (FIG. 21C), indicating an immunologically “cold” TME. Similarly, the frequency of CD8+GZMB+ CTLs (27.6±3.7% out of all CD8+ T cells) is the highest for the ICIE group (FIG. 21D & FIGS. 22E-22F), which is confirmed by immunofluorescence staining images (FIG. 21E and FIG. 22G) showing the distribution of CD8+GZMB+ cells in tumor sections. It is worth noting that unlike hyperthermic therapy for which permanent vascular stasis occurs due to heating, there is a temporary (a few hours) reperfusion of the tumor immediately after thawing a frozen tumor iceball, which may allow efficient immune cell infiltration into the tumor after cryosurgery to boost the antitumor responses. Collectively, these data support that the ICIE treatment may activate a strong antitumor immunity to kill cancer cells through a high level of GZMB secreted by CTLs.
ICIE Generates Both Short-Term and Long-Term Memory Immune Effects
To explore whether the activation of immune responses in primary tumor generates memory immune effects, the amount of effector memory T (TEM, CD8+CD44+CD62L−) cells were measured first. The TEM cells circulate in the blood and are ready to exert direct and rapid cytotoxicity against any existing tumors. Consistent with the improved antitumor immune responses in primary tumors, ICIE treatment significantly augments the proportion of TEM cells to 25.8±1.3% from 3.9±1.0% for PBS without cryosurgery (FIG. 21F and FIG. 23A). Also, cryosurgery significantly increases the TEM cell proportion for all the formulations than their counterparts without cryosurgery (FIG. 21G). In addition, ICIE promotes the percentage of central memory T cells (TCM, CD8+CD44+CD62L+) in spleen to 45.3±1.4% from 19.5±0.7% for PBS without cryosurgery (FIGS. 23B-23D), and mice received combinational treatments of cryosurgery and aforementioned formulations show significantly higher frequency of T than those with no cryosurgery (FIG. 23D), suggesting the capability of generating long-term protection against tumor burdens, metastasis, or relapse.
ICIE Boosts the Antitumor Immune Responses in Blood and Distant Tumors
As CD8+ T cells activated by the hot TME in primary tumor need to go with blood to enter distant tumors, we studied the CD8+ T cells together with Treg cells in blood first. The ratio of CD8+ to Treg cells (CD8+/Treg), an important value that has been used to predict the antitumor immune response and treatment outcome, was therefore determined using whole blood samples. ICIE dramatically increases the CD8+/Treg ratio to 90.7±19.3 from 5.0±0.8 for control treatment of PBS without cryosurgery (FIGS. 21H-21I and FIGS. 23E-23G). Again, for all the formulations, cryosurgery significantly increases the CD8+/Treg ratio in blood. Importantly, the ratio of CD8+ to Treg cells for the ICE treatment is higher than that for all the other treatments including CPT CRNPs, siR CRNPs, and free CPT&siR either with or without cryosurgery. Notably, the subpopulation of Treg cells in blood is almost unidentifiable following the ICIE (i.e., CPT&siR CRNPs with cryosurgery) treatment (FIG. 21I), while the Treg subpopulation is evident for the treatment of CPT&siR CRNPs without cryosurgery (2.8±0.5%, FIG. 23G). These results suggest that the ICIE treatment in primary tumors is very promising in boosting the antitumor immune responses to distant tumors.
Indeed, the ICIE treatment (i.e., CPT&siR CRNPs with cryosurgery) on the primary tumors increases the CD8+/Treg ratio to 8.1±2.7 in distant (Dist) tumors (without freezing) in the same mice, which is 376.2-folds higher than that for the right-side (R) tumors of the PBS control from mice with no cryosurgery (FIG. 21J and FIGS. 24A-24D). In contrast, a significantly lower CD8+/Treg ratio (0.2) is observed in the distant tumors from mice treated with CPT&siR CRNPs without cryosurgery. Although it is true that cryosurgery enhances CD8+/Treg ratios for all the formulations studied (FIG. 21J), the increase in the ratio is the most evident for the two formulations with siR. This suggests not only the cold-triggered drug/gene release but also the cold-triggered endo/lysosomal escape, in addition to the freezing-induced immune responses, is crucial to render the ICIE treatment with the potent antitumor immune responses against both primary and distant tumors. Moreover, ICIE elevates the percentage of CD8+GZMB−1T cells in distant tumors to 30.2±2.8% (out of all CD8+ T cells, FIG. 21K and FIG. 24E), which is 3.6-folds higher than that (8.3±0.5%) for its non-cryosurgical counterpart (FIG. 24F). Immunofluorescence staining of the distant tumors further confirms the aforementioned observations, showing more accumulation of CD8+GZMB+ T cells in the ICIE than other groups (FIG. 21L and FIG. 24G). Taken together, these data suggest that ICIE is promising for destroying not only primary but also distant tumors, which is further confirmed by tumor growth studies as detailed below.
ICIE Effectively Inhibits the Growth of Both Primary and Distant Tumors
To evaluate the therapeutic efficacy of the aforementioned antitumor immune responses, the dual orthotopic tumor model formed by injecting EO771 cells into both the left-side (L tumors for mice with no cryosurgery and primary tumors for mice with cryosurgery) and right-side (R tumors for mice with no cryosurgery and distant tumors for mice with cryosurgery done on the primary tumors) abdominal mammary fat pads of C57BL/6 mice as schematically illustrated in FIGS. 16A-16B, was continuously observed for thirty (37) days. As shown in FIGS. 25A-25B and FIGS. 26A-26D, cryosurgery evidently decreases the growth of both primary and distant tumors from mice with cryosurgery on their primary tumors for all the formulations, compared to that of the L and R tumors from mice with no cryosurgery. In particular, the ICIE treatment (i.e., CPT&siR CRNPs with cryosurgery) is the most effective in either inhibiting the tumor growth or inducing complete tumor eradication among all the treatments (with or without cryosurgery). In addition, in both the primary (left) and distant (right) tumors, the treatment of CPT&siR CRNPs results in lower tumor volumes than other formulations (free CPT&siR, CPT CRNPs and siR CRNPs) in the presence and absence of cryosurgery (FIGS. 26A-26D). The therapeutic efficacy was further confirmed by tumor images and tumor weights obtained at the final day (day 37) of the in vivo study. Again, for all the formulations, cryosurgery evidently reduces tumor sizes (FIGS. 27A-27C) and the reduction is significant for all the formulations according to the data of tumor weights (FIGS. 25C-25D). Also, either with or without cryosurgery, more inhibition of tumor growth and less tumor weight are observable for the formulation of CPT&siR CRNPs than all the other formulations including free CPT&siR, CPT CRNPs and siR CRNPs.
To further investigate the ICIE-induced tumor destruction, we sectioned the primary tumors from mice with cryosurgery and L tumor from mice with no cryosurgery and stained them for Ki-67 (representing tumor cell proliferation) and CD31 (representing angiogenesis). Similar to the tumor inhibition results, cryosurgery evidently downregulates the expression of both Ki-67 (FIG. 28A) and CD31 (FIG. 28B) in tumors treated with all the formulations, with the ICIE treatment results in the most reduction. This indicates the strongest inhibition of both tumor cell proliferation and tumor vascularization with the ICIE treatment. The antitumor effect was further investigated using hematoxylin & eosin (H&E) staining, which shows that necrosis is the most extensive in both primary and distant tumors from the ICIE group among all the other groups either with or without cryosurgery (FIG. 25E).
It is worth noting that no significant change of body weight was observed for mice treated with all formulations either with or without cryosurgery (FIGS. 29A-29B). Also, H&E staining analyses show no significant damages in the major organs (heart, liver, spleen, lung, and kidney) harvested from mice sacrificed at the end of the in vivo study for all the treatments with therapeutic agents and/or cryosurgery, when compared to that of the PBS control group (FIG. 30, FIG. 31), suggesting the good biosafety of CRNPs. Furthermore, no significant changes in the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels can be detected in blood of mice after treatment with CPT CRNPs, siR CRNPs, or CPT&siR CRNPs when compared to PBS group (FIGS. 32A-32B), confirming that the CRNPs cause negligible damage to the liver although they do have high accumulation in the liver after i.v. injection (FIGS. 14D-14E). Again, the minimal release of the encapsulated drugs from the CRNPs in the liver where no cryosurgery is performed, contributes to the negligible liver toxicity of the three different CRNPs. Collectively, these results indicate that ICIE generates an antitumor immune response to effectively kill both primary and distant tumors with no evident systemic toxicity.
ICIE Generates Long-Term Antitumor Memory Immune Response Against Metastatic Tumors
To further evaluate the generation of long-term antitumor memory immune response by ICIE for preventing lung metastasis, Balb/c mice with 4T1 orthotopic tumors (OTs) were applied with different treatments including ICIE (FIG. 33A). After priming for ten (10) days, a lung metastatic tumor model was formed by i.v. injection of 4T1 cancer cells (1×105 cells/mouse) and the mice were further injected with the various formulations till death or termination of experiments (FIG. 33A). The results show that ICIE can not only significantly inhibit the growth of orthotopic 4T1 tumor and decrease the orthotopic tumor weight to 2.6-fold and 2.9-fold less than that of CPT&siR CRNPs without cryosurgery on orthotopic tumor and PBS group with cryosurgery on orthotopic tumor, respectively (FIG. 34A), but also reduce the metastatic tumor burden in lungs, resulting in the lowest weight of lungs (0.21±0.03 g, which is close to the weight of normal lung) amongst all treatment groups (FIG. 33B). As shown in the representative images of lungs collected at the end of the study (FIG. 33C), ICIE greatly reduces the formation of metastatic foci in the lungs: the number of metastatic foci for the ICIE are 3.2-fold and 3.6-fold less than that of CPT&siR CRNPs without cryosurgery on orthotopic tumor and PBS group with cryosurgery on orthotopic tumor, respectively (FIG. 33D). This is further confirmed by the H&E staining data of lungs (FIG. 33E), showing least metastasis and most alveolar areas after ICIE treatment when compared to all other treatment groups. The strong inhibition on lung metastasis can be ascribed to the generation of the effector memory T (TEM, CD8+CD44+CD62L−) cells: ICIE significantly increases the effector memory T cells by 2.2 and 2.5 folds compared with the CPT&siR CRNPs without cryosurgery on orthotopic tumor and PBS group with cryosurgery on orthotopic tumor, respectively (FIG. 33F and FIG. 34B). As a result, the survival rate is significantly improved to 73% for mice with the ICIE treatment, while the survival rate of mice for the treatments of CPT&siR CRNPs without cryosurgery on orthotopic tumor and PBS group with cryosurgery on orthotopic tumor is 50% and 47%, respectively (FIG. 35). These results support that ICIE can be applied to generate a durable long-term antitumor memory immune response to inhibit tumor metastasis and prolong overall survival.
EXPERIMENTAL METHODS
Animals and Ethics Statement
The female C57BL/6 wildtype (4-week-old) and C57BL/6 OT-I (4-week-old) mice were purchased from Charles River (Wilmington, MA, USA), and The Jackson Laboratory (Bar Harbor, ME, USA), respectively. The Balb/cJ mice (4-week-old) were ordered from The Jackson Laboratory. All the mice (three to five mice per cage) were housed in standard, infection-free housing room, with 12 h light: 12 h dark cycles, stable room temperature at ˜23° C. and humidity of ˜40% in the vivarium at the University of Maryland, College Park. All procedures and animal cares were in accordance with the animal protocol (#R-May-18-24) approved by the Institutional Animal Care and Use Committee (IACUC) at University of Maryland, College Park.
Materials
Butyl acrylate (BA), dioxane, 2,20-azobis(2-methylpropionitrile) (AIBN), tetrahydrofuran, poly (D, L-lactide-co-glycolide) (PLGA, lactide/glycolide: 75/25, Mw: 4,000-15,000), MISSION® siRNA Fluorescent Universal Negative Control #1 (Cyanine 5), and Pluronic F127 (PF-127), were purchased from Sigma (St. Louis, MO, USA). The phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was ordered from Echelon Biosciences (Salt Lake City, UT, USA). N-isopropylacrylamide (NIPAAm), poly (vinyl alcohol) (PVA, Mw: 30,000-70,000 Da), Silencer™ GFP (eGFP) siRNA, CellTracker™ Green CMFDA Dye, and diethyl ether, were purchased from Thermo Fisher scientific (Waltham, MA, USA). Pharmaceutical grade chitosan oligosaccharide (Mw=1.2 kDa, 95% deacetylation) was received from Zhejiang Golden Shell Biochemical Co. Ltd (Zhejiang, China). The chitosan-modified PF-127 (CS-PF-127) was synthesized and purified using previous reported method. Irinotecan (CPT) was purchased from Selleckchem (Houston, TX, USA). Anti-mouse/rat ki-67 antibody (14-5698-82, clone: SolA15, dilution of 1:200), Alexa Fluor™ 488 goat anti-mouse IgG (A11001, dilution of 1:500), Alexa Fluor™ 488 goat anti-rabbit IgG (A11008, dilution of 1:500), Alexa Fluor™ 568 goat anti-mouse IgG (A11004, dilution of 1:500), Alexa Fluor™ 568 goat anti-rabbit IgG (A11011, dilution of 1:500) was purchased from Invitrogen (Waltham, MA, USA). The PD-L1 silencing siRNA (Pdcd-1L1 siRNA (m), sc-39700) and anti-mouse PD-L1 antibody (sc-518027, clone: D8, dilution of 1:1,000) were ordered from Santa Cruz (Dallas, TX, USA). Anti-rabbit HMGB1 antibody (3935S, dilution of 1:1,000), anti-rabbit calreticulin antibody (2891S, dilution of 1:1,000), anti-rabbit HSP-70 antibody (4872S, dilution of 1:1,000), anti-rabbit HSP-90 antibody (4874S, dilution of 1:1,000), anti-rabbit GAPDH antibody (2118S, clone: 14C10, dilution of 1:1,000), horseradish peroxidase (HRP)-anti-rabbit IgG antibody (7074P2, dilution of 1:1,000) and HRP-anti-mouse IgG antibody (7076P2, dilution of 1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-mouse CD31 antibody (ab28364, dilution of 1:200) was ordered from Abcam (Cambridge, United Kingdom). The antibodies for flow cytometry including Brilliant Violet 650™ anti-mouse CD206 (MMR) (141723, clone: C068C2, dilution of 1:200), Brilliant Violet 510™ anti-mouse/human CD11b (101263, clone: M1/70, dilution of 1:200), Brilliant Violet 605™ anti-mouse CD45 (103155, clone: 30-F11, dilution of 1:200), Brilliant Violet 785™ anti-mouse F4/80 (123141, clone: BM8, dilution of 1:200), Alexa Fluor® 647 anti-mouse Ly-6C (128010, lone: HK1.4, dilution of 1:200), Alexa Fluor® 700 anti-mouse Ly-6G (127622, clone: 1A8, dilution of 1:200), APC anti-mouse CD62L (104412, clone: MEL-14, dilution of 1:200), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 (320014, clone: 150D, dilution of 1:200), PE anti-mouse/human CD44 (103008, clone: IM7, dilution of 1:200), Alexa Fluor® 700 anti-mouse CD3 (100216, clone: 17A2, dilution of 1:200), Pacific Blue™ anti-mouse CD4 (100428, clone: GK1.5, dilution of 1:200), APC/Cyanine7 anti-mouse CD8a (100714, clone: 53-6.7, dilution of 1:200), PE anti-mouse CD11c (117308, clone: N418, dilution of 1:200), FITC anti-mouse CD86 (105006, clone: GL-1, dilution of 1:200), APC anti-mouse H-2Kb bound to SIINFEKL (141606, clone: 25-D1.16, dilution of 1:200), FITC anti-human/mouse granzyme B (GZMB) (515403, clone: GB11, dilution of 1:200), and PE anti-mouse CD274 (124308, clone: MIH7, dilution of 1:200), were purchased from Biolegend (San Diego, CA, USA). All other chemicals were ordered from Sigma unless specifically indicated otherwise.
Cells
EO771 medullary breast adenocarcinoma cells (CRL-3461) and 4T1 cells (CRL-2539) were purchased from ATCC (Manassas, VA, USA). EO771-OVA cells and GFP+ EO771 cells were generated as previously reported. To generate the EO771-OVA cell line, EO771 cells were transduced with a lentiviral vector expressing chicken ovalbumin gene, and selected for stable expression colonies via puromycin (1 μg ml−1) resistance and OVA presentation levels. A stable cell colony with >50% of the cells positive for OVA presentation in flow cytometrical analysis was selected and propagated for further use. To generate the GFP EO771 cell line, EO771 cells were transduced with a lentiviral vector expressing EGFP. At forty eight hours (48 h) after infection, the cells were selected with puromycin (1 μg ml−1) for 7 days and GFP-positive cells were collected by cell sorting. A stable GFP+ cell line was selected and expanded for further use. EO771 cells and 4T1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Gaithersburg, MD, USA) and Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco), respectively, supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS) under 5% CO2 at thirty seven degrees Celsius (37° C.) in a humidified cell incubator. Bone marrow dendritic cells (BMDCs) were collected from femurs of 6-week-old C57BL/6 mice) and cultured in RPMI medium containing interleukin-4 (IL-4, 10 ng ml−1, Peprotech, Cranbury, NJ, USA) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng ml−1, Peprotech) for 7 days. To culture the CD8+ T cells, single cell suspension was harvested from spleens and inguinal lymph nodes of 6-week-old C57BL/6 OT-I mice) through mechanical dissociation, and then purified by CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The resultant CD8+ T cells were maintained in RPMI medium supplemented with 10% FBS, and 10 ng ml−1 IL-2 (Peprotech) under 5% CO2 at thirty seven degrees Celsius (37° C.) in a humidified cell incubator.
Synthesis and Characterization of pNIPAAm-BA
The pNIPAAm-BA polymers with different ratios of NIPAAm to BA were synthesized by controlling the copolymerization of NIPAAm and BA using a reported procedure. Briefly, for the polymerization of the ratio of 81:19 (NIPAAm: BA), 16.2 mmol (1.834 g) of NIPAAm, and 3.8 mmol (0.488 g) of BA were transferred into a flask and dissolved in 10 ml of dioxane. After purging the resultant solution with nitrogen gas for 30 min, 11.61 mg of AIBN dissolved in 5 ml of dioxane was added dropwise into the flask. The flask was then immersed into an oil-bath at seventy degrees Celsius (70° C.) with the solution in it being stirred at 400 rpm for 12 h under a nitrogen atmosphere. The polymerization was stopped by exposing the flask to air for cooling down to room temperature (approximately twenty two degrees Celsius (˜22° C.)). Afterwards, the resultant sample was precipitated in excessive amounts of cold diethyl ether and filtered through a filter paper (size: 9.0 cm, VWR, Radnor, PA, USA). After dissolving in tetrahydrofuran and precipitating in cold diethyl ether for three times, the synthesized polymers were dried overnight under vacuum. The same procedure was used for the polymerization of NIPAAm and BA at the 80:20, 82:18, and 83:17 ratios, aside from the differences in the NIPAAm: BA molar ratios to give a total amount of 20 mmol for both monomers.
To confirm successful synthesis of the pNIPAAm-BA polymers, 10 mg of each polymer was dissolved in deuterated chloroform (CDCl3) and analyzed by H-NMR with a Ultrashield Plus 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA). The gel permeation chromatography (GPC) experiments were performed with a Waters (Milford, MA, USA) GPC instrument equipped with a 2414 refractive index (RI) detector and Styragel columns (HR4:5 μm, 7.8×300 mm and HR5:5 μm, 7.8×300 mm) in the presence of tetrahydrofuran as the eluent at a flow rate of 1 ml min−1 at 35° C. Polystyrene standard kits (Mid-High MW, Waters) were used for calibrating and calculating the molecular weight of the polymers. The data was plotted with Origin 8.0 (OriginLab Corporation, MA, USA).
Measurement of LCST
To determine LCST of the pNIPAAm-BA polymer, microscopic images of the aqueous samples of the polymers during cooling on a Linkam (Waterfield, UK) temperature-controlled microscope stage were taken. Briefly, a polymer film was prepared by dissolving 10 mg of polymer in 1 ml of ethanol and adding to the Linkam quartz crucible sample holder (Waterfield, UK) for drying. Then 500 μl of deionized water (DW) was added into the crucible of the Linkam temperature-controlled microscope stage mounted on a Zeiss Axio Scope A1 microscope (Oberkochen, Germany). After loading the holder with the sample into the stage, the stage was held at ten degrees Celsius (10° C.) for five minutes (5 min) for temperature equilibration in the sample. Then, the sample was cooled from ten degrees Celsius (10° C.) to negative fifteen degrees Celsius (−15° C.) at a cooling rate of 0.5° C. min−1, during which the sample images were taken every 20 s. Grayscale intensity of the images were quantified using the Image Processing Toolbox in MATLAB 2020b (MathWorks Inc., Natick, MA, United States). Grayscale values of all images were normalized to that of the first image at ten degrees Celsius (10° C.) and plotted against the recorded temperature during cooling. The change in grayscale value indicates a change in transparency in the sample. The LCST was determined as the temperature at the point where the second derivative of the data (i.e., the image grayscale value versus temperature) was equal to zero, or the inflection point in the curve of the image grayscale value versus temperature. The data was plotted with Origin 8.0.
Synthesis of Nanoparticles
CPT&siR CRNPs were prepared by a double-emulsion (water-in-oil-in-water or W-O-W) method. Briefly, to prepare the first water phase, 1 mg ml−1 aqueous chitosan solution was made by dissolving chitosan in 25 mM sodium acetate (pH 5.0). A total of 200 μl of chitosan solution was added into 100 μl of aqueous sodium chloride (NaCl) solution (20 mg ml) and mixed. During mixing, 50 μl of siRNA (100 μM) was added dropwise into the mixture and incubated for one hour (1 h) at room temperature. For the oil phase, 15 mg of PLGA, 30 mg of pNIPAAm-BA, 10 mg of PF-127, 5 mg of DPPC, and 2.5 mg of CPT were dissolved in 2 ml of DCM. To form the first W-O emulsion, the first water phase containing the siRNA was added dropwise into the oil phase and emulsified at an amplitude of 10% for 1 min using a Branson 450 sonifier (Hampton, NH, USA). The first emulsion was then added into 5 ml of the second water phase, which was 2% PVA solution containing 10 mg of CS-PF127. The resultant sample was further emulsified by sonication (10%) for another 1 min to form the W-O-W emulsion, which was stirred overnight at 1200 rpm using a stir bar. Right after sonication, 5 ml of 2% PVA solution was added into the second emulsion. The sample was centrifuged (13800 g) at room temperature for twenty minutes (20 min) to collect the CPT&siR CRNPs, which were washed with DW once and redispersed in DW. The procedures for making the CPT CRNPs, siR CRNPs, and CPT&siR PLGA nanoparticles were the same as that for making the CPT&siR CRNPs except that siR, CPT, and pNIPAAm-BA were not used, respectively. For the preparation of CPT&siR CRNPs (no NaCl), the procedure was the same with that for preparing the CPT&siR CRNPs except that NaCl was not used.
Characterization of Nanoparticles
To determine the encapsulation efficiency (EE) and loading capacity (LC) of CPT, the pellets of the nanoparticles were dissolved in DMSO. The absorbance of CPT was measured by a Tecan (Männedorf, Switzerland) Spark Multimode Microplate Reader at 375 nm and the amount of drug was quantified by a calibration curve made with various known concentrations of CPT. EE and LC were calculated as: EE=(CPT in the pellet)/(CPT fed for synthesis)×100%, LC=(CPT in the pellet)/(Total weight of the pellet)×100%. For quantifying the amount of siR in the nanoparticles, siR was isolated by dissolving the pellets with chloroform and extracting the siR with diethylpyrocarbonate (DEPC)-treated tris(hydroxymethyl) aminomethane-ethylenediaminetetraacetic acid (TE) buffer. The concentration of isolated siR was analyzed by NanoDrop 2000c (Thermo Fisher Scientific).
The phase transition of the CPT&siR PLGA NPs and CPT&siR CRNPs was examined by immersing their aqueous samples in sealed glass vials into cold NaCl solution (negative four degrees Celsius (−4° C.)) for ten minutes (10 min), followed by gradually warming them up to room temperature. Visual appearances of the samples during the cooling-warming treatment were studied by taking their photographs. The LCST of nanoparticles in aqueous sample was also determined by the Linkam temperature-controlled microscope stage in the same way as mentioned above for the pNIPAAm-BA polymers.
The hydrodynamic diameter, zeta potential, and polydispersity index (PDI) of the nanoparticles in DW at a concentration of 0.1 mg ml−1 before and after cold treatment were measured by dynamic light scattering (DLS) using a Malvern (Cambridge, UK) Zetasizer Nano ZS instrument. The data of hydrodynamic diameter were plotted with Origin 8.0. The morphology of these nanoparticles with/without cold treatment was also examined by JEOL (Akishima, Tokyo, Japan) JEM 2100 LaB6 TEM after staining with uranyl acetate solution (2%, w w−1) and Hitachi (Chiyoda City, Tokyo, Japan) SU-70 Schottky field emission gun scanning electron microscope after drying in the chemical hood. Moreover, the stability of CPT&siR CRNPs in PBS (0.1 mg ml−1) were examined by measuring the hydrodynamic diameter and PDI for 5 days.
The stability of siR in CPT&siR CRNPs (1.0 mg ml−1) after incubation with PBS for zero hours (0 h), four hours (4 h), eight hours (8 h), twelve hours (12 h), and twenty four hours (24 h) were measured by gel retardation assay using 3.0% agarose gel comprising GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). The gel retardation assay was performed in 1× Tris-Borate-EDTA (TBE, Fisher Scientific, Hampton, NH, USA) running buffer at 120 v for fifteen minutes (15 min) using a Bio-Rad (Hercules, CA) Horizontal Electrophoresis Systems.
Characterization of Cold-Triggered Drug/Gene Release
To assess the in vitro cold-triggered drug release profile, aliquots of 10 mg of CPT&Cy5-siR CRNPs were dispersed in 3 ml of acetate buffer (pH 5.0), phosphate buffer (pH 6.5), and phosphate buffer (pH 7.4) in a 15-ml centrifuge tube, respectively, and then placed in an Incubating Orbital Shaker (VWR, Radnor, PA, USA) at one hundred revolutions per minute (100 rpm) and thirty seven degrees Celsius (37° C.). For cold treatment at eight hours (8 h), the samples were immersed into cold NaCl solution (negative four degrees Celsius (−4° C.)) for ten minutes (10 min). At various times, 400 μl of the supernatant of the samples were collected for release measurement after centrifuging the samples at room temperature or 0° C. (only for the time point right after the cold treatment) at 13800 g for twenty minutes (20 min). A total of 400 μl of fresh buffer was added into the sample right after each collection of the supernatant. The fluorescence intensity of CPT in the collected supernatant was measured using the Spark Multimode Microplate Reader, with an excitation wavelength at 370 nm and an emission wavelength at 434 nm. The amount of Cy5-siR released at different time points was also quantified by the Spark Multimode Microplate Reader with an excitation wavelength at 651 nm and an emission wavelength at 670 nm. The cold-triggered release of siR from the nanoparticles at negative four degrees Celsius (−4° C.), twenty two degrees Celsius (22° C.), and thirty seven degrees Celsius (37° C.) were also investigated with the aforementioned gel retardation assay.
In Vitro Cellular Uptake and Intracellular Distribution
For the cellular uptake study, GFP+ EO771 cells and normal lymphocytes from mouse spleen were seeded on collagen-coated coverslips in 12-well plate or 12-well plate only at a density of 1×105 cells per well and incubated for 12 h. After removing the culture medium, cells were treated with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs at thirty seven degrees Celsius (37° C.) for eight hours (8 h). The concentrations of CPT and Cy5-siR were 10 μg ml−1 and 50 nM, respectively. For confocal analysis, the treated cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) for fifteen minutes (15 min) at room temperature, and stained with 6-diamidino-2-phenylindole (DAPI, 300 nM) for ten minutes (10 min) at room temperature. Before imaging with a Zeiss LSM 710 confocal microscope, the samples were mounted on glass slides with the mounting medium (CC/Mount™, Sigma-Aldrich). The images were collected with the Zeiss ZEN 2011 SP7 FP3 (black) and analyzed with the Zeiss ZEN 2.6 lite. To quantify cell uptake, the treated cells were detached with trypsin, fixed with 4% PFA, and analyzed by a BD (Franklin Lakes, NJ, USA) FACSCelesta flow cytometer with the built-in software BD FACSDiva™ 8.0.1.1 for data collection.
To visualize the intracellular distribution, EO771 cells grown on collagen-coated coverslips as aforementioned were incubated with PBS, free CPT&Cy5-siR, and CPT&Cy5-siR CRNPs (without or with cold treatment) at the same concentration of Cy5-siR (50 nM) for up to eight hours (8 h). At eight hours (8 h), cells for the cold treatment group (CPT&Cy5-siR CRNPs+C) was washed with PBS, replaced with fresh medium, and transported into the chamber of a SP (Gardiner, NY, USA) VirTis AdVantage Pro Freeze Dryer/Lyophilizer, where the temperature was maintained at negative four degrees Celsius (−4° C.). After ten minutes (10 min), the samples were taken out of the chamber and warmed back passively to room temperature in a biosafety cabinet. Before mounting to glass slides for imaging by the confocal microscope, the samples were stained with 50 nM LysoTracker™ Green DND-26 (Invitrogen), fixed with 4% PFA, and stained with DAPI. To determine the intracellular distribution of CPT&Cy5-siR CRNPs (no NaCl) in the presence or absence of cold treatment, same procedures were performed except for substituting the CPT&Cy5-siR CRNPs with CPT&Cy5-siR CRNPs (no NaCl).
In Vitro Gene Silencing
To investigate gene silencing, GFP EO771 cells were seeded on collagen-coated coverslips in a 6-well plate at a density of 1×105 cells per well. After overnight incubation, cells were treated with PBS, free GFP-siR, GFP-siR CRNPs, GFP-siR CRNPs+C, and Scrambled (Sc)-siR CRNPs+C at the same concentration of GFP-siR (50 nM). At eight hours (8 h), cells for the groups with cold treatment were placed into the negative four degrees Celsius (−4° C.) chamber as mentioned above and incubated at negative four degrees Celsius (−4° C.) for ten minutes (10 min) before warming up to room temperature and putting back in the incubator. After further incubation for 40 h, cells were collected and analyzed by flow cytometry or confocal microscopy. For the latter, cells were washed with PBS three times and stained with 50 nM LysoTracker™ Red DND-99 (Invitrogen) and DAPI using the same staining method as described above. Furthermore, the same procedure was used for investigating the downregulation of PD-L1 expression, except substituting the GFP-siR with PD-L1-siRNA (siR) and the cells were stained with PE anti-mouse CD274 at cold (ice-water) temperature for twenty minutes (20 min) before flow cytometry analysis.
In Vitro Anticancer Effect
Viability of EO771 cells after treatment with blank CRNPs, free CPT&siR, CPT&siR CRNPs, and CPT&siR CRNPs+C at increasing amounts of CPT ranging from 0 to 40 μg ml−1 was examined using Cell Counting Kit-8 (CCK-8, Dojindo, Rockwill, MD, USA) assay. The siR concentration was kept at 50 nM. Briefly, EO771 cells were seeded in 96-well plates at a density of 2×104 cells per well and cultured for 12 h. The cells were treated with the various formulations and incubated for twenty four hours (24 h). For the cold treatment, after incubation with the CPT&siR CRNPs for eight hours (8 h), the plate was placed into the negative four degrees Celsius (−4° C.) chamber and maintained at negative four degrees Celsius (−4° C.) for ten minutes (10 min) before warming up to room temperature and putting it back to the incubator. At twenty four hours (24 h), cells were washed with PBS and supplemented with 100 μl of fresh medium. Then a ten microliters (10 μl) of CCK-8 was added into each well and incubated for one hour (1 h). Absorbance was then measured using the Spark Multimode Microplate Reader at 460 nm. To determine cell apoptosis and necrosis, EO771 cells (2×105 cells per well) were seeded in 6-well plates. After overnight incubation, the cells were treated with PBS, free CPT&siR, CPT&siR CRNPs, or CPT&siR CRNPs+C. The CPT and siR concentrations were ten micrograms per microliter (10 μg ml−1) and 50 nM, respectively. For the cold treatment, it was done at eight hours (8 h) as aforementioned. At twenty four hours (24 h), cells were collected and stained with a FITC-Annexin V Apoptosis Detection Kit I (BD) according to the protocol provided by the manufacturer. The samples were analyzed by flow cytometry (BD).
Measurement of ICD In Vitro
To determine the generation of ICD, expression of DAMP molecules including HMGB1, CRT, HSP-70, and HSP-90 was investigated by flow cytometry. Briefly, EO771 cells (2×105 cells per well) were seeded in 6-well plates and incubated overnight. The medium was changed to a fresh medium containing PBS, free CPT&siR, CPT CRNPs, siR CRNPs, or CPT&siR CRNPs. The CPT and siR concentrations were ten micrograms per microliter (10 μg ml−1) and 50 nM, respectively. After eight hours (8 h), cells for the groups with cold treatment were placed into a cold chamber maintained at negative four degrees Celsius (−4° C.) or negative twenty degrees Celsius (−20° C.) for ten minutes (10 min). After warming back to room temperature, the cells were put back in the incubator for further culture. At twenty four hours (24 h), cells were collected, fixed with 4% PFA for ten minutes (10 min), and incubated with primary antibodies of CRT, HMGB1, HSP-70 and HSP-90 overnight. After washing with PBS and further incubated with corresponding fluorescence-tagged secondary antibodies, all the samples were analyzed by flow cytometry.
In Vitro Maturation of BMDCs and Activation of CD8+ T Cells
To investigate the maturation of BMDCs, EO771-OVA cells (1×105) were seeded on the top wells (i.e., inserts) of transwells and treated with PBS, free CPT&siR, CPT&siR CRNPs and CPT&siR CRNPs+C for eight hours (8 h). The CPT and siR concentrations were ten micrograms per microliter (10 μg ml−1) and 50 nM, respectively, and the cold treatment at negative twenty degrees Celsius (−20° C.) for ten minutes (10 min) was done at eight hours (8 h) as aforementioned. Afterward, immature BMDCs were seeded in the bottom wells of the transwells at 1×106 cells per well. At twenty four hours (24 h), the BMDCs were collected and stained with Alexa Fluor® 700 anti-mouse CD3 (for T cell exclusion), PE anti-mouse CD11c, FITC anti-mouse CD86, and APC anti-mouse H-2Kb (bound to SIINFEKL) antibodies for analyzing with flow cytometry. The co-culture medium was also collected to examine the secretion of IFN-γ and TNF-α by ELISA kits (Biolegend) following the protocols provided by the manufacturer.
To determine T cell proliferation, OT-I CD8+ T cells (1×105) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer's instruction and co-cultured for three (3) days with BMDCs (1×105) pre-incubated with EO771-OVA cells with the various treatments for twenty four hours (24 h) given above in 96-well plate. Proliferation of CD8+ T cells was measured by flow cytometry. For the detection of T cell activation, non-labeled OT-I CD8+ T cells (1×105) were co-cultured with the aforementioned pre-incubated BMDCs (1×105) in 96-well plate. After three (3) days, cells were stimulated with Cell Stimulation Cocktail (eBioscience, San Diego, CA, USA) for four hours (4 h), stained with Alexa Fluor® 700 anti-mouse CD3, APC/Cyanine7 anti-mouse CD8a, and FITC anti-human/mouse GZMB antibodies, and analyzed by flow cytometry.
In Vitro Antitumor Capability
EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye (0.5 μM) were seeded in μ-Dish35 mm, low (ibidi, Fitchburg, WI, USA) at a density of 1×104 cells per dish and placed in the cell-incubator stage (mounted on the Zeiss LSM710 microscope) maintained at thirty seven degrees Celsius (37° C.) with 5% CO2. The CD8+ T cells (2×103 per dish) activated as mentioned above were added into the dishes with EO771-OVA cells and monitored for 4 h under the microscope. At the end of the study, cells were harvested, stained with propidium iodide (PI), and further analyzed by flow cytometry.
In Vivo Biodistribution of CRNPs
The in vivo distribution of CRNPs was investigated in C57BL/6 mice bearing EO771 orthotopic tumors. Briefly, 1×106 of EO771 cells per mouse were administered into the left abdominal mammary fat pad of C57BL/6 mice. When the volume of tumor reached approximately 100 mm3, mice were randomly distributed into three groups and intravenously injected with PBS, free CPT&Cy5-siR, or CPT&Cy5-siR CRNPs. The doses of CPT and Cy5-siR were 2.0 mg kg−1 and 30 μg kg−1, respectively. At zero hours (0 h), four hours (4 h), eight hours (8 h), twelve hours (12 h), and twenty four hours (24 h), whole-animal images of all mice were acquired using a PerkinElmer (Waltham, MA, USA) IVIS instrument. Moreover, circulation of CRNPs in blood was also studied by collecting blood from the mice at various time points and imaged by the IVIS system. At the end of the study, mice were euthanized and their critical organs including hearts, livers, spleens, lungs, kidneys, and tumors were harvested and imaged by IVIS.
In Vitro Hemolysis Assay
Whole blood collected from C57BL/6 mice was centrifuged at 500 g and washed with PBS for 3 times. A total of 0.5 ml of whole blood cells (4% in PBS) were mixed with 0.5 ml of DW, PBS, or CPT&siR CRNPs at various NP concentrations from 25 to 800 μg ml−1. After incubation for twenty four hours (24 h) at thirty seven degrees Celsius (37° C.), the samples were centrifuged at 500 g for five minutes (5 min). Supernatants were collected and their absorbances at 540 nm were measured by the Spark Multimode Microplate Reader. In addition, after dehydration by a series of ethanol solutions (50%, 75%, 85%, 95%, and 100%), the morphology of erythrocytes after incubation with CPT&siR CRNPs at 800 μg ml−1 was compared to that from the PBS group via taking their SEM images using the Hitachi SU-70 Schottky field emission gun scanning electron microscope by following the procedures provided by the manufacturer.
In Vivo Antitumor Capability Through Cryo-Immunotherapy
To evaluate the antitumor cryo-immunotherapy effect, 1×106 of EO771 cells per gland were administered into both the left and right abdominal mammary fat pad of C57BL/6 mice. On day 10, mice with EO771 tumors at both sides were randomly divided into ten groups (five groups without cryosurgery/cryo and five groups with cryosurgery done on the primary tumors on the left side of the mice) and intravenously administered with PBS (no cryosurgery/Cryo), free CPT&siR (no Cryo), CPT CRNPs (no Cryo), siR CRNPs (no Cryo), and CPT&siR CRNPs (no cryo); and PBS (Cryo), free CPT&siR (Cryo), CPT CRNPs (Cryo), siR CRNPs (Cryo), CPT&siR CRNPs (Cryo). The number (n) of mice for each group is 9 with 6 mice for monitoring the tumor growth and 3 mice for analyzing the immune cells and cytokines on day 20. The doses of CPT and Cy5-siR were 2.0 mg kg−1 and 30 μg kg−1, respectively. Cryosurgery was done on the left tumors at eight hours (8 h) after the first injection of the various formulations. The injections were continued every three (3) days until the end of the study. Body weight and tumor size were recorded every three (3) days, and tumor volume was calculated as follows: V=0.5×L×W2, where V, L, and W are the tumor volume, long diameter, and short diameter, respectively. The maximal tumor size/burden permitted by the IACUC at the University of Maryland, College Park, is 1.5 cm in long/short diameter. During the whole experiment, the maximal tumor size/burden was not exceeded. For cryosurgery, mice were anesthetized with isoflurane first. Cryosurgery was then performed by touching the skin of the tumor area with a cryoprobe (Dahai Mechanical & Electronic Equipment Manufactory, Jiangsu, China) at its central location of the tumor area and freezing for a total of ten minutes (10 min). The cryoprobe was connected to a DH-286 Cryogenic Therapeutic Apparatus (Dahai Mechanical & Electronic Equipment Manufactory). Two K-type thermocouples were placed at the center and boundary of the tumor area for monitoring the temperature at the two locations, and the freezing was turned on and off to maintain the temperature at tumor center and boundary at approximately negative twenty degrees Celsius (−20° C.) and negative four degrees Celsius (−4° C.), respectively.
On day 20, 3 mice per groups were euthanized for harvesting tumors and blood for further analysis. Briefly, the obtained tumors were minced into pieces and digested with DNAse I (0.1 mg ml−1, Roche, Basel, Switzerland) and collagenase IV (2.0 mg ml−1, Roche) for one hour (1 h) at thirty seven degrees Celsius (37° C.). Before analyzing by flow cytometry, the single cell suspensions were filtered through a 70-μm strainer and stained and gated with a combination of markers: dendritic cells (CD3−CD11c+CD86), Treg cells (CD3+CD4+Foxp3+), activated CD8 (CD3+CD8+GZMB+), tumor associated macrophage (CD45+CD11b+Ly6C−Ly6G−F4/80+) M1 (F4/80+CD206−CD86+), tumor associated macrophage M2 (F4/80+CD206+CD86−), monocytic myeloid-derived suppressor cells (M-MDSC, CD11b+Ly6C+Ly6G−), and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC, CD11b+Ly6C−Ly6G+). The examination of immune cells in blood was performed by removing the erythrocytes with RBC lysis buffer (Biolegend) and staining with antibodies similarly as mentioned above. For analysis of memory immune response, the collected blood and spleen cells were stained and gated with a combination of markers for CD8+ effector memory T (TEM, CD3+CD4−CD8+CD62L−CD44+) cells and central memory T (TCM, CD8+CD44+CD62L+) cells. All flow cytometry data was analyzed with FlowJo (v10, BD). Moreover, after cryosection, the presence of CD8+GZMB+ cells and the generation of HMGB1, CRT, HSP-70 and HSP-90 in tumor tissues were investigated by immunofluorescence staining. Briefly, cryosectioned tumor slides were washed with PBS for 3 times, fixed with 4% PFA for ten minutes (10 min), and rinsed with PBS for 3 times. Afterward, the samples were permeabilized with 0.2% Triton X-100 for five minutes (5 min), washed with PBS for 3 times, and incubated with 5% BSA for one hour (1 h) at room temperature. After removing the BSA with 3 times of PBS washing, the samples were incubated with the primary antibodies overnight at 4° C. After 3 times of PBS washing, the samples were incubated with the corresponding fluorescence-labeled secondary antibodies for 30 min at room temperature. The samples were further stained with DAPI, washed with PBS, and mounted with the coverslips for imaging with the Zeiss LSM 710 microscope. Furthermore, the expression of PD-L1 in in vivo tumors after various treatments was determined by western blotting. Briefly, tumor tissues were weighted, minced, and lysed with 1× radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Cell Signaling Technology). Supernatants of samples were collected after centrifuging at 13,200 g and 4° C. for 30 min. The total proteins were separated with 10% SDS-PAGE gel and then transferred onto the polyvinylidene difluoride (PVDF) membrane (0.45 μm, MilliporeSigma, Burlington, MA, USA). After blocking with 5% bovine serum albumin (BSA) and washing using phosphate buffered saline with Tween 20 (PBST), the membranes were incubated with the primary antibodies of PD-L1 overnight. Afterwards, the membranes were incubated with corresponding HRP-conjugated secondary antibodies for one hour (1 h), immersed in Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), and scanned by the FluorChem E System Gel Imaging System (ProteinSimple, San Jose, CA, USA).
At the end of the study, mice were euthanized and critical organs including hearts, livers, spleens, lungs, kidneys, and tumors were harvested from the mice. Tumors were weighted. H&E staining was conducted for paraffin-sectioned slices of all the organs by following the standard procedure. Tumors were also cryosectioned by following the standard procedure for immunostaining of CD31 and Ki-67. The amount of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in blood serum of mice collected at the end of the study were also measured by Mouse ALT ELISA Kit (Abcam) and Mouse AST ELISA Kit (Abcam) using the protocols provided by the manufacturer.
In Vivo Anti-Metastatic Effect of ICIE
To examine the long-term antitumor memory immune response of ICIE, Balb/c mice were inoculated with 4T1 cells into the mammary fat pad of mice at 1×106 cells per gland. After 5 days, mice with orthotopic 4T1 tumors were randomly grouped and injected with various formulations including: PBS, PBS +cryo, CPT&siR CRNPs, and CPT&siR CRNPs+cryo. The number of mice in each group is 8, with another group of 8 mice named PBS without orthotopic tumor as control for further metastatic induction. The doses of CPT and Cy5-siR were 2.0 mg kg−1 and 30 μg kg−1, respectively. Cryosurgery was done on the orthotopic tumors at eight hours (8 h) after the first injection of the various formulations. All injections were continued every three (3) days until the death of the animals or the end of the study. To establish the in vivo lung metastatic model, each Balb/c mouse was intravenously injected with 1×105 4T1 cells 10 days post the first injection of different formulations. After monitoring for 15 days, all live mice were euthanized for collection of tumors and metastatic lungs. H&E staining was conducted for paraffin-sectioned slices of lungs by following the standard procedure.
Statistical Analysis
All quantitative data are presented as mean±standard deviation (S.D.) from three or more independent runs. One-way analysis of variance (ANOVA) with Tukey's post-test and correction for multiple comparisons was used when multiple groups with one independent variable/factor (e.g., treatment) were compared. Two-way ANOVA with Sidak's post-test and correction for multiple comparisons were used when multiple groups with two independent variables/factors (e.g., treatment and dose). Survival benefit was analyzed by Log-rank (Mantel-Cox) test. Statistical analysis and plotting were carried out by using the GraphPad (San Diego, CA, USA) Prism 9 software, unless it is specifically mentioned otherwise above (i.e., plotted with Origin 8.0). A p value less than 0.05 indicates statistically significant difference.
From the foregoing, it can be seen that the present disclosure accomplishes at least all of the stated objectives.
The “invention” is not intended to refer to any single embodiment of the particular invention but encompass all possible embodiments as described in the specification and the claims. The “scope” of the present disclosure is defined by the appended claims, along with the full scope of equivalents to which such claims are entitled. The scope of the disclosure is further qualified as including any possible modification to any of the aspects and/or embodiments disclosed herein which would result in other embodiments, combinations, subcombinations, or the like that would be obvious to those skilled in the art.
Patent Application
20240238027
