Patent Application
20240042054
The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 12, 2023, is named “Replacement_Seq_List-WIS0070US2” and is 3,686 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.
The present disclosure is related to hydrogel compositions loaded with at least two types of particles, and methods of using the loaded hydrogels to treat primary and inoperable tumors through pyroptosis.
Pyroptosis, a type of programmed cell death mediated by gasdermin proteins, is characterized by the continuous expansion of cells forming large ballooning bubbles until the cell membrane ruptures, resulting in the release of cellular contents and subsequent activation of a strong inflammatory response. As an important innate immune response in the body, pyroptosis plays a crucial role in antagonizing infection and endogenous danger signals. Moreover, the latest research reveals that cytotoxic lymphocytes rely on gasdermin-mediated pyroptosis to kill tumor cells, suggesting that pyroptosis is also closely involved in anti-cancer immune response and rising as a very promising method for cancer treatment. Specifically, granzyme A released from cytotoxic lymphocytes cleaves gasdermin B (GSDMB) to trigger pyroptosis in target tumor cells, while cytotoxic lymphocytes secreted granzyme B or chemotherapy induced activated caspase 3 cleaves gasdermin E (GSDME) to trigger tumor pyroptosis. In addition, it is believed that activated caspase 8 could cleave gasdermin C (GSDMC) to trigger tumor pyroptosis. Moreover, the working mechanism of gasdermin A (GSDMA) in tumor pyroptosis is unclear, but it was found that GSDMA could also trigger tumor pyroptosis and exhibit antitumor efficacy.
What is needed are improved compositions and methods to stimulate pyroptosis.
In an aspect, a composition comprises a hydrogel, the hydrogel loaded with a) bacterial particles comprising a gasdermin D (GSDMD) protein cage conjugated to a surface thereof, and b) nanoparticles loaded with an ESCRT inhibitor. The hydrogel can be an injectable hydrogel or a thermosensitive hydrogel.
In another aspect, a method of treating a primary or metastatic tumor comprises locally injecting the injectable hydrogel described above at the site of the primary or metastatic tumor.
In a further aspect, a method of treating an inoperable cancer comprises implanting the thermosensitive hydrogel described above at the site of the inoperable cancer.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
Among all gasdermin family proteins, membrane perforin gasdermin D (GSDMD) acts as an effective executor of pyroptosis mainly in immune cells. Notably, there is increasing evidence showing that pyroptosis can inhibit cancer cell proliferation by inducing inflammatory cell death. Leveraging pyroptosis-mediated anti-tumor immune response to overcome the immunosuppression is a novel strategy for cancer immunotherapy.
Despite the encouraging progress of pyroptosis-mediated cancer immunotherapy, there are still emerging limitations restricting its wider application. First, the expression of gasdermins in cancer tissue is suppressed by DNA methylation. For example, methylation of DFNA5 (deafness autosomal dominant 5) gene reduced the GSDME expression in most tumor tissues, thus restricting T cell- or NK cell-mediated cytotoxicity to tumor cells. Furthermore, even if there is the expression of full-length gasdermins in tumor cells, pyroptosis cannot occur when the associated caspase signaling pathway is not activated in the tumor cells. More importantly, calcium influx-triggered assembly of the endosomal sorting complexes required for transport (ESCRT) III system could prevent the cell from programmed cell death and further cell lysis through facilitating damaged cell membrane repair, which significantly dampened the tumor pyroptosis. Therefore, providing sufficient gasdermin intracellular delivery, activating the related caspase signaling pathway and preventing ESCRT mediated cell membrane repair are progressive prerequisites for the realization and enhancement of pyroptosis-mediated cancer immunotherapy.
To leverage gasdermin-triggered pyroptosis for anti-tumor immunotherapy, described herein is an intracellular bacterium-attenuated Salmonella typhimurium (VNP) delivery system to shuttle gasdermin D protein to initiate the tumor cell pyroptosis. To facilitate the VNP modification and further intracellular release of GSDMD, GSDMD proteins are assembled into protein nanocages by crosslinking proteins through bifunctional linkers such as glutathione (GSH)-responsive linkers. GSDMD protein cage-conjugated VNP bacteria (designated VNP-GD) could effectively shuttle GSDMD to the cytoplasm of the tumor cells, which will subsequently release GSDMD intracellularly upon the activation of elevated GSH concentration to trigger tumor cell pyroptosis (
The hydrogel delivery system described herein comprises two types of particles. The first particles include gasdermin D (GSDMD), specifically bacterial particles comprising a GSDMD protein cage conjugated to a surface thereof and the second particles include an endosomal sorting complex required for transport (ESCRT) inhibitor.
The first particles include gasdermin D (GSDMD). As used herein, GSDMD is an executor of pyroptosis in immune cells. While the experiments herein were performed with mouse GSDMSD (SEQ ID NO: 1), the disclosure also include human GSDMSD (SEQ ID NO: 2; accession number NP_001159709), and GSDMSD from other mammalian species. Recombinant GSDMD of SEQ ID NO: 1 is commercially available from MyBioSource.com.
In an aspect, the GSDMD is in the form of a protein cage. As used herein, a protein cage is a hollow, typically spherical, typically monodisperse, three-dimensional crosslinked structure which facilitates conjugation of the GSDMD to the surface of a bacteria particle. Exemplary protein cages have diameters of 24.4 to 396 nm, specifically about 130 nm.
The GSDMD protein cages may be prepared by crosslinking GSDMD with a bifunctional linker such as a glutathione (GSH)-responsive linker, which can react with the amino group in the protein and release the protein under GSH trigger. Excess GSH linkers may be removed by ultrafiltration. Furthermore, the protein cages are conjugated on the surface of a bacteria by amide reaction of carboxyl groups in the protein cages and amino groups on the bacteria, and the GSH-responsive linker provides release of the GSDMD protein from the bacteria particle without affecting the activity and structure of the protein when the intracellular GSH causes cleavage of the linker.
Additional bifunctional linkers include reactive oxygen species (ROS)-responsive linkers, pH-responsive linkers, enzyme-responsive linkers, and the like. Exemplary ROS-responsive linkers include thioether-containing polymers, diselenide, thioketal, arylboronic ester, aminoacrylate, oligoproline, peroxalate ester, and mesoporous silicon. Exemplary pH-responsive linkers include pH-sensitive hydrazone linkers, orthoester linkers, weak acid labile linkers, and the like. Exemplary enzyme-responsive linkers include matrix metalloproteinase (MMP)-degradable peptide linkers.
Bacteria such as Salmonella, Clostridium and Bifidobacterium have a natural tropism for solid tumors, and that this tropism may be exploited to facilitate the selective delivery of therapeutic agents to tumor cells. The bacteria particle thus shuttles the GSDMD protein cages to the cytoplasm of the tumor cells. Exemplary bacteria particles include an attenuated Salmonella typhimurium (VNP) delivery system, such as VNP-20009 and Clostridium, Bifidobacterium, Listeria, Streptococcus and Escherichia coli-based delivery systems, such as ECN1917. VNP-20009 (Vion Pharmaceuticals Inc) was created by the chromosomal deletion of two genes, purl (purine biosynthesis) and msbB (LPS biosynthesis) and was attenuated by at least 10,000-fold in mice compared with the parental wild-type strain.
The second particles include an endosomal sorting complex required for transport (ESCRT) inhibitor. Without being held to theory, it is believed that endosomal sorting complexes required for transport (ESCRT) III-mediated cell membrane repair significantly diminishes the tumor cell pyroptosis through forming microvesicles and subsequently removing gasdermin pores from the plasma membrane. The ESCRT inhibitor overcomes the plasma membrane repair mediated by ESCRT III machinery. Exemplary ESCRT inhibitors include the Ca2+ chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)), FGI-104 (4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-6-[4-(hydroxymethyl)-3-methoxyphenyl]phenol), and RNA interference-mediated inhibitors. RNA interference-mediated inhibitors can be used to deplete ESCART proteins such as ESCRT 0, I, II and III.
To facilitate delivery of the ESCRT inhibitor, the ESCRT inhibitor is encapsulated in a biodegradable nanoparticle such as a dextran nanoparticle to facilitate sustained release.
As used herein, “biodegradable” materials are those that, when introduced into cells, are broken down by cellular machinery (e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain aspects, components generated by the breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo. Biodegradable materials may be enzymatically broken down, or broken down by hydrolysis, for example, into their component polymers. Breakdown of biodegradable materials (including, for example, biodegradable polymeric materials) may include hydrolysis of ester bonds, cleavage of urethane linkages, and the like.
Exemplary materials for biodegradable nanoparticles include poly-lactic acid (PLA); poly-D-L-glycolide (PLG); poly-D-L-lactide-co-glycolide (PLGA), poly-alkyl-cyanoacrylate (PCA), poly-ε-caprolactone, gelatin, alginate, chitosan, agarose, polysaccharides, and proteins. Biodegradable nanoparticles can be made by techniques known in the art such as solvent evaporation, spontaneous emulsification, nanoprecipitation, salting out, polymerization, or ionic gelation of hydrophilic polymers, for example.
In an aspect, the biodegradable nanoparticle comprises a polysaccharide. The term “polysaccharide” refers to a polymer of sugars. Polysaccharide nanoparticles described herein may be made of polysaccharides such as dextran, amylose, amylopectin, glycogen, cellulose, arabonixylan, and/or pectin. In certain embodiments, the polysaccharide is dextran. Dextran is a complex, branched glucan (a polysaccharide made of many glucose molecules) composed of chains of varying lengths (from 3-2000 kilodaltons). The straight-chain comprises alpha-1,6-glycosidic linkages between glucose molecules, while branching begins at alpha-1,3 linkages. In some embodiments, dextran nanoparticles are comprised of carboxymethyl dextran.
The polysaccharides that make up the nanoparticles can have a range of molecular weights such as 1 kDa to 1 million kDa (e.g., 1-10 kDa, 10-100 kDa, 100-1000 kDa, or 1000-1,000,000 kDa). The polysaccharide nanoparticles can have an average diameter in a range of 1 nm-500 nm (e.g., 1-10 nm, 10-25 nm, 25-50 nm, 50-100 nm, or 100-500 nm). The polysaccharide nanoparticles may be relatively monodisperse (e.g., diameters of particles all within a range of 10 nm or less of each other) or more polydisperse.
The hydrogel of the delivery system is a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. “Biocompatible hydrogel refers” to a hydrogel that is not toxic to living cells.
Examples of materials that can be used to form a biocompatible hydrogel include polysaccharides such as alginate, polyphosphazines, poly(acrylic acids), poly(methacrylic acids), poly(alkylene oxides), poly(vinyl acetate), polyvinylpyrrolidone (PVP), and copolymers and blends. Additional materials for forming hydrogels include agarose, alginic acid, chitosan, dextran, dextran sulfate, heparan, heparan sulfate, cellulose sulphate, carrageenan, gellan gum, xanthan gum, guar gum, chondroitin sulfate, hyaluronic acid, collagen, gelatin, hydroxyethyl starch and poly(N-isopropyl acrylamide). Combinations of the foregoing materials may be employed.
In an aspect, the hydrogel is a thermosensitive hydrogel. Exemplary materials to form thermosensitive hydrogels include polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymers such as Pluronic® F127 and F108, which are PEO-PPO block copolymers with molecular weights of 12,600 and 14,600, respectively. Each of these compounds is available from BASF (Mount Olive, N.J.). Pluronic® F108 at can form a thermosensitive hydrogel at a concentration of 20-28% in phosphate buffered saline (PBS). Pluronic® F127 at a 20-35% concentration in PBS also forms thermosensitive hydrogels. When the hydrogel is a thermosensitive hydrogel, the composition can be in the form of a hydrogel patch. Advantageously, hydrogel patches can be implanted or placed adjacent to a solid tumor to stimulate pyroptosis and treat cancer.
In another aspect, the hydrogel is an injectable hydrogel. Injectable hydrogels can include polysaccharides such as heparan, heparan sulfate, chitosan, hyaluronic acid, dextran, alginic acid and hydroxyethyl starch. A crosslinker such as a reactive polyethylene glycol crosslinker can be used to form the hydrogel. An exemplary injectable hydrogel is a hyaluronic acid hydrogel, formed by adding Extralink®-Lite (polyethylene glycol diacrylate (PEGDA)) was added to a Glycosil® (thiol-modified hyaluronan). Injectable hydrogels are locally injectable to release therapeutic agents at the site of a solid tumor.
In an aspect, the hydrogel composition further comprises an immune checkpoint inhibitor. When the hydrogel comprising first and second particles is combined with immune checkpoint inhibitors, the tumor immunotherapy efficacy has been substantially augmented, effectively inhibiting tumor growth and prolonging the survival of tumor-bearing mice. Exemplary immune checkpoint inhibitors include peptides with high affinity for an immune checkpoint receptor such as PD-L1, PD-1, OX40, TIGIT, CTLA-4, CD137 (4-1BB), CD28, and CD27.
In an aspect, a method of treating a primary tumor comprises locally injecting the injectable hydrogel described herein at the site of the primary or metastatic tumor. In an aspect, the primary tumor is a metastatic breast cancer tumor, a melanoma, sarcoma, prostate cancer, cervical cancer, and the like. The 4T1 tumor model with lung metastasis is an established mouse model for the lung metastasis of 4T1 breast cancer. The B16F10 tumor model is a B16F10 tumor model is a murine melanoma cell line. In an aspect, the composition further comprises an immune checkpoint inhibitor, such as a peptide with high affinity for an immune checkpoint receptor, wherein the immune checkpoint receptor comprises PD-L1, PD-1, OX40, TIGIT, CTLA-4, CD137 (4-1BB), CD28, and CD27.
In another aspect, a method of treating an inoperable cancer comprises implanting the thermosensitive hydrogel described herein at the site of the inoperable cancer. Exemplary inoperable cancers include inoperable ovarian cancer, lung cancer, pancreatic cancer, liver cancer, and colorectal cancer. The ID8-Luc tumor model is a mouse model for orthotopic ovarian cancer. In an aspect, the composition further comprises an immune checkpoint inhibitor, such as a peptide with high affinity for an immune checkpoint receptor, wherein the immune checkpoint receptor comprises PD-L1, PD-1, OX40, TIGIT, CTLA-4, CD137 (4-1BB), CD28, and CD27.
The invention is further illustrated by the following non-limiting examples.
Cells and antibodies: The murine 4T1, B16F10, ID8 cell lines and VNP20009 were purchased from ATCC. Luciferase-expressed B16F10 and 4T1 were obtained from Imanis Life Sciences Inc. Luciferase-expressed ID8 cells were provided by Dr. Paula Hammond's lab at MIT. Cells were cultured in the CO2 incubator (Fisher) at 37° C. with 5% CO2 and 90% relative humidity. The antibodies used in this study were summarized as follows (company, clone, category number): GoInVivo™ Purified anti-mouse CD279 (PD-1) (BioLegend, RMP1-14, 114114), Fluorescein isothiocyanate (FITC)-anti-mouse CD45 (BioLegend, 30-F11, 103108), APC-anti-mouse CD3 (BioLegend, 17A2, 100236), FITC-anti-mouse CD4 (BioLegend, GK1.5, 100406), PE-anti-mouse CD8a (BioLegend, 53-6.7, 100708), FITC-anti-mouse IFNγ (BioLegend, XMG1.2, 505806), PerCP/Cy5.5-anti-human/mouse Granzyme B (BioLegend, QA16A02, 372212), PE-anti-mouse CD45 (BioLegend, 30-F11, 103106), Alexa Fluor® 594 anti-mouse CD8a (BioLegend, 53-6.7, 100758), FITC-anti-mouse CD11c (BioLegend, N418, 117306), PE-anti-mouse CD80 (BioLegend, 16-10A1, 104708), APC-anti-mouse CD86 (BioLegend, GL-1, 105012), Precision Count Beads (BioLegend, 424902). All antibody dilutions were performed following the manufacture's guidance (diluted by approximately 200 times for use).
Synthesis of the GSH responsive linker: 2-hydroxyethyl disulphide (R, 200 mg, 1.30 mmol) was dissolved in the anhydrous acetonitrile (ACN, 12 mL). N, N′-disuccinimidyl carbonate (DSC, 1.33 g, 5.19 mmol) and Et3N (1.05 ml, 7.79 mmol) were added. The mixture was stirred for 8 hours at room temperature under nitrogen protection, followed by the removal of the solvent under vacuum. The crude product was then dissolved in CH2Cl2 (20 mL). The solution was washed with saturated NaHCO3 solution, saturated NH4Cl solution, brine in sequence, and dried over anhydrous Na2SO4. The solvent in the organic phase was evaporated, and the solid that remained was recrystallized with ethyl acetate (20 ml). The resulting white solid was dried under vacuum (RS, 380 mg, yield 65%). 1H-NMR (CDCl3, 300 MHz): δ (ppm) 4.60 (t, 4H), 3.07 (t, 4H), 2.86 (s, 8H). ESI (m/z): calcd for C14H16N2O10S2, 436.4 [M]; found, 459.0 [M+Na]+.
Synthesis, preparation, and characterization of ESCRT inhibitor-loaded dextran nanoparticles: Dextran (1.0 g, Mn approximately 9-11 kDa) dissolved in 10 ml was added to a flame-dried round-bottom flask, then pyridinium P-toluenesulfonate (PPTS, 15.6 mg, 0.062 mmol) and 2-ethoxypropene (4.16 mL, 37 mmol) were added during stirring. The reaction was stirred at room temperature for 30 min and quenched by adding 1 mL of triethylamine. The precipitated mixture was washed three times in basic water (pH approximately 8) to prevent undesired degradation, centrifugated (8000 rpm, 15 min), and lyophilized to obtain the final white solid powder (m-dextran). To prepare the ESCRT inhibitor BAPTA-AM-loaded dextran nanoparticle, 10 mg m-dextran and 0.5 mg BAPTA-AM were dissolved in 2 mL dichloromethane (DCM). Then, 4 mL 3% poly (vinyl alcohol) (PVA) solution was added, and sonication (2 min in total, 2s on, 2s off, 40% power, ice bath) was performed. Next, the mixture was dispersed in 20 ml 0.3% PVA solution in the beaker and stirred for 1 hour. The emulsion was centrifuged at 14,000 rpm for 35 min to collect the dextran nanoparticles (EI-NP). The supernatant was removed, and EI-NP was dispersed in 1 ml PBS. The particle size and zeta potential were measured with the Malvern Zetasizer instrument. The morphology of EI-NP was observed under Transmission Electron Microscope (TEM) imaging. To investigate the release profile of the ESCRT inhibitor, the EI-NP suspended in 2 ml PBS with 0.1% Tween 80 was loaded in a 20,000 MWCO dialysis cassette (Thermo scientific) for the analysis of drug release by high-performance liquid chromatography (HPLC) at different time points.
Preparation and characterization of the protein cage-conjugated bacteria: To prepare the protein cages, the GSDMD proteins (MyBioSource, 0.0143 μmol) were dissolved in the PBS solution (132 μl, PH 7.4), then the GSH responsive linkers (0.214 μmol) dissolved in the DMSO (9.35 μl) were added and stirred at room temperature for 35 min to form the protein cage. The particle size and zeta potential of the protein cage were measured by the Malvern Zetasizer instrument. The morphology of the protein cages was observed under TEM imaging. Next, for the conjugation of protein cages on the surface of bacteria, the obtained protein cages and EDC/NHS (0.214 μmol) were added into the VNP20009 suspension (108 CFU-ml−1) and incubated for one hour in a 37° C. shaker incubator at 250 rpm. The reaction mixture was centrifuged (4,000 rpm, 5 min) to obtain the protein cage-conjugated bacteria (VNP-GD). The morphology of VNP-GD was observed under TEM imaging.
To verify the successful conjugation of the protein cage on the surface of the bacteria, the protein was labeled with Rhodamine B and formed into protein cages before being conjugated onto the bacteria. Centrifugation (4,000 rpm) was performed to remove the unconjugated protein in the supernatant. Then the bacteria were labeled with Hoechst 33342 for 15 min at room temperature and washed with PBS three times before confocal imaging. The release of proteins from the protein cage-conjugated bacteria with or without the trigger of GSH (10 mM) was further characterized by measuring the protein concentration in the supernatant after centrifugation (4,000 rpm) at different time points.
Characterization and verification of VNP-GD induced tumor cell pyroptosis: 2×105 4T1 cells were seeded into the confocal dishes, incubated overnight, and then treated with PBS, VNP, GD (GSDMD protein cage), VNP-GD (GSDMD protein cage-conjugated VNP), and VNP-GD+EI-NP (GSDMD protein cage-conjugated VNP+EI-NP) for 24 hours (GSDMD=2 μM, EI=4 μM, VNP=106 CFU mL−1). After washing with PBS, the morphology of cells was observed under a confocal microscope. In addition, the Annexin V (staining at room temperature for 15 min) was used to label the cell membrane after pyroptosis. To further verify pyroptosis-mediated pore formation, 2×105 4T1 cells were seeded into the 24-well plate, incubated overnight, and then treated with PBS, VNP, GD, VNP-GD, and VNP-GD+EI-NP for 24 hours (GSDMD=2 μM, EI=4 μM, VNP=106 CFU mL−1). SYTOX@ green and PI staining were performed respectively, and the cell uptake of the SYTOX@ green and PI were detected by the flow cytometry. Moreover, the LDH release was detected by the Invitrogen™ CyQUANT™ LDH Cytotoxicity Assay Kit according to the instruction and operation manual. HMGB1 expression was detected by the HMGB1 ELISA Kit accordingly.
Western blot assay of the pyroptosis related signaling pathway: 5×105 4T1 cells were seeded into 6-well plate, incubated overnight, and then treated with PBS, VNP, GD, VNP-GD, and VNP-G+EI-NP for 24 hours (GSDMD=2 μM, EI=4 μM, VNP=106 CFU mL−1). After washing with PBS, the cells were digested with trypsin and collected into 1.5 ml tubes. Centrifugation (1,000 rpm, 4 min) was performed, and 100 μl Pierce™ RIPA buffer with protease inhibitor cocktail was added to each tube for 1-hour cell lysis with vortex every 10 min. Then, the lysed cells were centrifuged under 14,000 rpm for 30 min. The supernatant was collected, and BCA analysis was performed to determine the protein concentration. The protein samples added with protein loading buffer were boiled for 15 min at 100° C. water bath and stored at −20° C. for further use.
To perform the western blot assay, the 1× running buffer was prepared and gel plate was placed into the gel holder. Then the running buffer was added, and the protein marker and various samples were loaded accordingly. SDS-PAGE was performed at 120 V until the bromophenol blue indicator ran to the bottom. The proteins in the gels were further transferred onto a PVDF membrane (350 mA, 85 min). After incubation with 5% skim milk for 2 hours at room temperature, the PVDF membranes were incubated with the primary rabbit antibodies (anti-Gasdermin D, anti-Cleaved Caspase 1, anti-Cleaved Gasdermin D, anti-HMGB1 and anti-β actin, respectively) at 4° C. overnight. The membranes were then washed with TBST buffer three times (each time 10 min) and incubated with the secondary antibody (Anti-rabbit IgG, HRP-linked) at room temperature for 1 hour. After washing with TBST another three times, Western Blot Substrates were added to the membrane, and western blot images were taken by the iBright™ Imaging Systems.
Flow cytometry assay of calcium influx and the confocal imaging of ESCRT III-mediated cell membrane repair during pyroptosis: For the flow cytometry assay of calcium influx, 2×105 4T1 cells were seeded into the 24-well plates, incubated overnight, and then treated with PBS, VNP, GD, VNP-GD, and VNP-GD+EI-NP for 24 hours (GSDMD=2 μM, EI=4 μM, VNP=106 CFU mL−1). Cells were washed twice with PBS and incubated with 4 μM Fluo-8 for 45 min in the dark. Then cells were washed twice with PBS and digested with trypsin, and the fluorescence intensity of Fluo-8 was detected by the flow cytometry. For the confocal imaging of ESCRT III-mediated cell membrane repair, first, the plasmid containing the CHMP3-mCherry sequence was constructed by Addgene and extracted with the Monarch@ Plasmid Miniprep Kit according to the manufacture instructions. The plasmid concentration was measured with a microplate reader, and the plasmids were stored at −20° C. for further use. To obtain 4T1 cells expressing CHMP3-mCherry, 2×105 4T1 cells were seeded into the 24-well plates and incubated overnight. Then, 2.5 μg CHMP3-Mcherry DNA plasmid and P 3000™ Regent were mixed in 125 μl DEME medium without FBS in a 1.5 ml tube (A). Meantime, 7.5 μl Lipofectamine™ 3000 was dissolved in 125 μl DEME medium without FBS in a 1.5 ml tube (B). Then the mixture in tube A was added to tube B and mixed well by pipetting and incubated for 10 min at room temperature. After washing with PBS, 600 μl DEME medium (with 10% FBS) was added to the 4T1 cell-containing confocal dishes. The final mixture in tube B was added slowly and evenly into the confocal dishes containing 4T1 cells. After 36 hours, the expression of CHMP3-mCherry was verified by the confocal imaging. Then 4T1 cells expressing the CHMP3-mCherry were further treated with VNP-GD and VNP-GD+EI-NP for 24 hours (GSDMD=2 μM, EI=4 μM, VNP=106 CFU mL−1). After washing with PBS, the cell membrane was labeled with Annexin V in binding buffer for 15 min under room temperature, and the confocal imaging was performed to observe the ESCRT III-mediated cell membrane repair.
Hydrogel preparation and bacteria release from the hydrogel: To prepare the Pluronic® F127 thermosensitive hydrogel, 2 g Pluronic® F127 was dissolved into 10 ml PBS solution under room temperature to form the 20% hydrogel solution. The gelation time of the 20% Pluronic® F127 hydrogel was measured by incubating the hydrogel at 37° C. incubator, which formed into gel in 60-70s. The injectable Pluronic® F127 hydrogel delivery system was used for the treatment of 4T1 breast cancer model and B16F10 melanoma tumor model. To prepare the hyaluronic acid hydrogel, the Extralink®-Lite (PEGDA) was added to a mixture of Glycosil® (thiol-modified hyaluronan loaded with the bacteria delivery system) at 1:4 ratio, and it took about 30 min for the gelation. Then the bacteria release assay was performed after loading 107 CFU bacteria into the hydrogel. The hydrogel loaded with bacteria was loaded in a cell strainer placed on a 6-well plate, and then the 6-well plate was submerged with PBS. At different time points, 100 μl of PBS solution in the plate was collected, diluted, and spread on LB medium plate with bacterial spreader. The number of released bacteria was calculated by counting the number of the formed clones accordingly. For the ID8 ovarian cancer treatment, the prepared EI-NP was loaded into hyaluronic acid hydrogel and lyophilization was performed to obtain the off the shelf hydrogel patch. VNP-GD was loaded into the hydrogel patch to form the final therapeutic cell patch before implantation. As for the stability assay, the particle size of EI-NP after lyophilization was measured at predetermined time points with the Malvern Zetasizer instrument.
In vivo anti-tumor efficacy: The BALB/c (Female, aged 6-8 weeks) and C57BL/6 mice (Male, aged 6-8 weeks) were purchased from Jackson laboratory. The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison. Mice were euthanized at humane endpoints if any of the following criteria were met: (i) weight loss or gain of >20%, (ii) moribund, (iii) severe abdominal swelling, (iv) jaundice, or (v) tumor volume >2000 mm3. To verify the anti-tumor efficacy of our treatment strategies, we first established the 4T1 breast cancer model by implanting 4T1 cells in the breast pad of the BALB/c mice. Seven days later, different formulations were loaded into the Pluronic® F127 hydrogel for peritumoral injection, including PBS, VNP@Gel (hydrogel loaded with VNP), GD/EI-NP@Gel (GSDMD protein cage and EI-NP co-loaded in the hydrogel), VNP-GD@Gel (GSDMD protein cage-conjugated VNP loaded in the hydrogel), VNP-GD/EI-NP@Gel (GSDMD protein cage-conjugated VNP and EI-NP co-loaded in the hydrogel) and VNP-GD/EI-NP@Gel+aPD-1 (GSDMD protein cage-conjugated VNP and EI-NP co-loaded in the hydrogel with systemic injection of aPD-1 antibodies). GSDMD=2 mg/kg, EI=5 mg/kg, VNP=107 CFU per mouse, aPD-1=2.5 mg/kg (three doses on day 0, day 2 and day 4). The tumor volume was measured and calculated based on the equation: length×width2×0.5. The survival of the mice was monitored accordingly. Next, a breast cancer lung metastasis model was established to evaluate the anti-metastasis effect of the hydrogel-based delivery systems. Briefly, 4T1 cells were injected into the breast pad of BALB/c mice on day −7, different treatments were administered by peritumoral injection after 7 days, including PBS, VNP@Gel, GD/EI-NP@Gel, VNP-GD@Gel, VNP-GD/EI-NP@Gel and VNP-GD/EI-NP@Gel+aPD-1. GSDMD=2 mg/kg, EI=5 mg/kg, VNP=107 CFU per mouse, aPD-1=2.5 mg/kg (three doses on day 0, day 2 and day 4). Then, on day 7, 2×105 4T1 cells were administered to the mice in different treatment groups by i.v. injection through tail vein. On day 21, the mice were euthanized, and the lungs were collected for further analysis. The lungs were washed with saline and fixed with Bouin's solution for 6 hours, and pictures were taken to observe the surface lung metastasis nodules. Furthermore, H&E assays were performed to observe the 4T1 tumor lung metastasis.
To further verify the anti-tumor efficacy of the hydrogel-based delivery systems, a melanoma tumor model was established by injecting B16F10 cells into the right flank of C57BL/6 mice on day −7. Seven days later, different formulations were loaded into the Pluronic® F127 hydrogel for peritumoral injection, including PBS, VNP@Gel, GD/EI-NP@Gel, VNP-GD@Gel, VNP-GD/EI-NP@Gel and VNP-GD/EI-NP@Gel+aPD-1. GSDMD=2 mg/kg, EI=5 mg/kg, VNP=107 CFU per mouse, aPD-1=2.5 mg/kg (three doses on day 0, day 2 and day 4). The tumor volume was measured and calculated using the equation: length×width2×0.5. The survival of the mice was monitored accordingly. To verify if the local treatment strategy could activate the systemic immunity to inhibit the growth of the distant tumor, a double B16F10 tumor model was established. Briefly, on day −7, the primary B16F10 tumor was established by injecting the B16F10 cells into the right flank of the C57BL/6 mice. Six days later, the second tumor was established by injecting B16F10 cells on the left flank of the C57BL/6 mice. The tumor volume of the second tumor was monitored according to the equation: length×width2×0.5.
To extend our developed technology for treating inoperable tumors, an advanced ovarian tumor model was established. Briefly, 1×107 ID8-Luc ovarian tumor cells were injected intraperitoneally into six-week-old female C57BL/6 mice. One week later, the establishment of the ovarian tumor model was verified by the IVIS imaging system. D-luciferin potassium salt was dissolved into PBS and intraperitoneally injected into the mice (100 μl, 3 mg D-luciferin potassium salt per mouse). Five minutes after the injection, bioluminescence imaging was performed to record the distribution and growth of the ID8-Luc cells in the enterocoelia. After the establishment of the inoperable ovarian tumor model, surgery was performed to open the abdominal cavity of the mice, and different hydrogel-based delivery systems including PBS, VNP@Gel, GD/EI-NP@Gel, VNP-GD@Gel, VNP-GD/EI-NP@Gel, VNP-GD/EI-NP@Gel+aPD-1 and VNP-GD/EI-NP@Patch+aPD1 (GSDMD protein cage-conjugated VNP and EI-NP loaded in the cell patch with three times systemic injection of aPD-1) were implanted accordingly. GSDMD=2 mg/kg, EI=5 mg/kg, VNP=107 CFU per mouse, aPD-1=2.5 mg/kg (three doses on day 0, day 2 and day 4). IVIS imaging was performed at predetermined time points to monitor the tumor growth.
In vivo immune activation: To verify the immune activation of our treatment strategies, we established the 4T1 breast cancer model by implanting 4T1 cells in the breast pad of the BALB/c mice. Seven days later, different formulations were loaded into the Pluronic® F127 hydrogel for peritumoral injection, including PBS, VNP@Gel, GD/EI-NP@Gel, VNP-GD@Gel, VNP-GD/EI-NP@Gel and VNP-GD/EI-NP@Gel+aPD-1. GSDMD=2 mg/kg, EI=5 mg/kg, VNP=107 CFU per mouse, aPD-1=2.5 mg/kg (three doses on day 0, day 2 and day 4). Then one week later, the tumors and lymph nodes were harvested, weighed, washed with PBS, cut into small pieces, and digested with DEME medium containing 0.5 mg/ml collagenase for 1 hour at a 37° C. incubator. After the digested tumor tissues were mechanically disrupted, they were filtered through a 40 μm cell strainer. For the analysis of lymph nodes, the cell suspension was stained with anti-mouse CD11c, anti-mouse CD80, and anti-mouse CD86. For the analysis of tumor tissue, the cell suspension was stained with anti-mouse CD3, anti-mouse CD4, anti-mouse CD8a, and anti-mouse Granzyme B antibodies and analyzed using flow cytometry with Attune™ NxT Flow Cytometer software (All these antibodies were diluted by ˜200 times). The cytokines expressions in the tumor tissue after different treatments were analyzed by the LEGENDplex™ Multi-Analyte Flow Assay Kit, and IFNγ and TNFα ELISA kits according to the manufacture's guidance.
Statistics: All the results are shown as mean±s.d. The GraphPad Prism software was used to perform statistical analysis, and analysis of variance (ANOVA) was used to compare multiple groups (>two groups) statistically. Log-rank test was performed for the statistical analysis of the survival study. A P value lower than 0.05 (*P<0.05) was considered the threshold for statistical significance among control groups and experimental groups.
Cells tend to maintain redox homeostasis; the oxidative stress in tumor tissue usually causes higher glutathione (GSH) expression in tumor cells than that in normal cells. Therefore, to achieve selective protein release inside tumor cells, a GSH responsive linker (
Under normal circumstances, the Ca2+ concentration outside the cell is much higher than the intracellular Ca2+ concentration, and the maintenance of this concentration gradient is mainly governed by the calcium ion channel on the cell surface. However, after pyroptosis-induced pore formation and cell membrane damage, the calcium influx would trigger the ESCRT-mediated cell membrane repair. To inhibit the calcium influx triggered ESCRT cell membrane repair for improving VNP-GD-induced tumor cell pyroptosis, a biocompatible dextran nanoparticle (EI-NP) loaded with the ESCRT inhibitor (a potent calcium ion antagonist, BAPTA-AM) was formulated, showing the monodispersed spherical structure under TEM observation with a particle size of approximately 150 nm and the zeta potential of −1.8 mV (
The good biosafety and tumor-targeting tendency of VNP makes it an excellent vector to deliver therapeutic proteins directly to tumors. As an intracellular bacteria strain, VNP efficiently delivered GD protein cages into 4T1 cells (
SYTOX® green is a nucleic acid dye that can be applied to characterize the cell membrane integrity since it can only pass through damaged cytoplasmic membranes to bind the nucleic acid in which its fluorescence intensity will be significantly enhanced after binding. Therefore, the cell uptake of SYTOX® green could serve as an indication of tumor cell pyroptosis after different treatments. It was found that the VNP-GD increased the cell uptake of SYTOX green from 3.8% (control group) to 30%. Notably, VNP-GD+EI-NP treatment significantly enhanced the cell uptake of SYTOX green to 52%, suggesting the strongest pyroptosis-induced cell membrane rupture and pore formation among all treatment groups (
GSDMD protein-induced pyroptosis signaling pathway is mainly illuminated in immune cells and remains elusive in tumor cells. To reveal the underlying mechanism of VNP-GD-triggered tumor cell pyroptosis, the western blot assay was first performed to investigate the key protein expressions in the activated tumor cell pyroptosis signaling pathway. As shown in
To investigate the EI-NP-mediated pyroptosis enhancement, the ESCRT III machinery-mediated membrane repair was investigated after VNP-GD+EI-NP treatment. The calcium influx, the initiator of ESCRT III-induced membrane repair, was first evaluated to verify if pyroptosis could trigger calcium influx and if EI-NP could inhibit the calcium influx in the 4T1 tumor cells. As shown in
The in vivo pyroptosis-triggered tumor immunotherapy efficacy of VNP-GD and EI-NP was first evaluated on a 4T1 breast cancer model (
As shown in
Moreover, to validate the broad applicability of the VNP-GD/EI-NP@Gel+aPD-1 treatment strategy, a B16F10 melanoma tumor model was established and treated with various formulations (
Pyroptosis is a form of programmed cell death that produces a large number of inflammatory factors, releases tumor antigens, and initiates antigen-presenting cells (APC)-mediated adaptive immune responses, which could be leveraged to reawaken the immune system and overcome the tumor immunosuppressive microenvironment. Therefore, the dendritic cell (DC) maturation was first investigated after different treatments, and it was found that VNP-GD@Gel increased the CD80+CD86+ proportion of DCs from 12.9% (PBS group) to 28.7%, while VNP-GD/EI-NP@Gel increased the DC maturation to 49.4%, significantly higher than that of VNP-GD@Gel group (
Furthermore, according to the ELISA assay of HMGB1 expression in
Ovarian cancer is a highly malignant tumor, and because of the lack of typical clinical symptoms in the early stage, many patients are associated with a large number of organ metastases in the abdominal cavity, including metastases in the liver, spleen, and kidney when diagnosed, which makes surgical treatment impossible. Therefore, in view of this clinical challenge, for inoperable tumor treatment, such as advanced metastatic ovarian cancer, a pyroptosis-enhancing cell patch was prepared with a lyophilized hyaluronan hydrogel loaded with EI-NP (
Pyroptosis, an inflammatory cell necrosis that has evolved from bacterial infection-triggered caspase-1 dependent cell death to gasdermin-dependent programmed cell death, has attracted extensive attention from basic research to disease treatment. For instance, inhibiting immune cell pyroptosis can be utilized to treat inflammatory diseases such as cardiovascular disease and sepsis, while promoting cell pyroptosis can be used for anti-tumor immunotherapy. However, for tumor cell pyroptosis, overexpression of DNA methyltransferase restricted the expression of gasdermins, especially GSDME (DFNA5), prevents the application of leveraging gasdermin-dependent tumor cell pyroptosis for anti-tumor treatment. To overcome this, the integration of DNA methyltransferase inhibitors that can increase the GSDME expression with chemical drugs that can activate the caspase-3 pathway to cleave GSDME for perforating membrane, has been suggested for triggering GSDME-mediated tumor pyroptosis. Despite GSDME protein, other gasdermin family proteins, including GSDMA, GSDMB, GSDMC, and GSDMD, have been found to trigger cell pyroptosis through an activated pore-forming domain unleashed by the distinct enzyme initiator. Even though there are extensive mechanistic investigations, few have entered in vivo studies, particularly for anti-tumor applications. The reasons accounting for the lack of in vivo anti-tumor evaluations include the relatively restricted Gasdermin protein expression in tumor cells, the complication of initiating gasdermin-mediated tumor cell pyroptosis requiring activation of multiple signaling pathways to unleash the pore-forming domain of Gasdermin protein, and the presence of ESCRT III machinery-dependent membrane repair, which work synergistically inhibit the in vivo applications of gasdermin-dependent pyroptosis.
Described herein is a VNP bacteria-based GSDMD protein delivery system for in vivo anti-tumor treatment, in which VNP could efficiently shuttle GSDMD to the intracellular compartment of tumor cells where flagella on VNP activated capapase-1 to further cleave delivered GSDMD to N-terminal domain for effective pore-forming mediated pyroptosis. To overcome ESCRT III machinery-induced cell membrane repair that can compromise the efficacy of tumor cell pyroptosis, a biodegradable nanoparticle was prepared to load Ca2+ chelator that can inhibit calcium influx to prevent the recruitment of ESCRT III machinery. Through the combination of VNP-mediated delivery and activation of GSDMD and inhibition of ESCRT III-dependent membrane repair, in vivo anti-tumor treatment efficacy has been demonstrated on multiple tumor models.
Free GSDMD is difficult to diffuse into tumor cells due to their relatively large molecular weight and negative surface charge, and the full length of GSDMD cannot trigger the cell pyroptosis due to the concealment of their active pore-forming domain. To address this challenge, leveraging the superior tumor targeting ability of bacteria delivery systems, we constructed a GSDMD protein cage (GD) with a GSH-responsive linker and further decorated GD on the surface of attenuated Salmonella typhimurium (VNP) to form VNP-GD. As described herein, GSDMD proteins could be efficiently transported to the cytoplasm of tumor cells, in which the VNP could trigger the activation of cleaved caspase 1 to transform intracellularly delivered GSDMD into pore-forming domain N-terminal GSDMD for binding to the plasma membrane and subsequently triggering pyroptosis in tumor cells. Furthermore, ESCRT III mediated membrane repair induced by the calcium influx during pyroptosis could help the tumor cells survive and increase the resistance of tumor cells to pyroptosis. Specifically, after the gasdermin-NT induced pore formation, calcium influx through the pore would trigger ESCRT-mediated membrane repair to subsequently form macrovesicle and stop the continuous expansion of cells and the release of inside antigens, diminishing the treatment efficacy of tumor pyroptosis. To address this issue, described herein is a biocompatible nanoparticle (EI-NP) to bioresponsively release Ca2+ chelator to inhibit calcium influx and subsequently prevent ESCRT-dependent membrane repair. In vitro the combination of VNP-GD and EI-NP worked synergistically to initiate and further strengthen the tumor cell pyroptosis, as evidenced by large ballooning bubbles formed and increased release of intracellular contents in both 4T1 and B16F10 cells. To enable in vivo anti-tumor applications, two distinct formulations (an injectable hydrogel and a lyophilized cell patch) were developed for treating multiple tumor settings. In the 4T1 and B16F10 tumor model, the VNP-GD/EI-NP@Gel elicited a strong local and systemic anti-tumor immune response that inhibits the primary and distant tumor growth and prevents the tumor metastasis. Further synergistic treatment efficacy was demonstrated when combining VNP-GD/EI-NP@Gel with systemic injection of immune checkpoint inhibitors, which was probably due to the enhanced tumor antigen release triggered by tumor pyroptosis leading to more T cell infiltration and activation that can be further strengthened through blocking PD1/PDL1 pathway. To treat the clinically inoperable tumor such as late-stage ovarian cancer, a VNP-GD/EI-NP@Gel cell patch was designed and implanted into the abdominal cavity of mice, which significantly inhibited the tumor growth and spread and prolonged the survival time of the tumor-bearing mice.
In summary, described herein is a hydrogel-based delivery system serving as a local reservoir to sustainedly release VNP-GD and EI-NP for enhanced programmed tumor cell death by integrating VNP-activated GSDMD-dependent tumor pyroptosis and inhibition of ESCRT III-mediated plasma membrane repair.
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. “About” or “approximately” as used herein is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ±10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims priority to U.S. Provisional Application 63/390,444 filed on Jul. 19, 2022, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63390444 | Jul 2022 | US |
