Patent Application
20220370777
This patent document relates to devices, systems and methods for in situ delivery of cargo payload to a biological organism or tissue, for example, via a degradable microneedle.
Multiple methods have been proposed for the delivery of a wide variety of payloads for transdermal delivery, including diffusion transport, encapsulation in nano-microparticles, as well as enhanced delivery using external fields such as temperature.
The use of microneedles has facilitated the painless and localized delivery of drugs across the skin. Although their use has successfully addressed the treatment of multiple diseases, the efficacy of these devices has been limited by the slow diffusion of drug through the skin or the requirement of external triggers to achieve a faster active delivery. For example, although microneedles have been a very successful platform for the delivery of these active compounds, the main disadvantages of conventional approaches are: (1) a two-step administration process is typically needed; (2) not having morphology such as an insufficiently sharp tip (suffering decreased penetration ability); and (3) requiring an efficient coating procedure. Common diffusion technology incorporated into microneedle patches provide a well-established platform and commodity to users. However, diffusion alone presents a linear release kinetic behavior and may not be sufficient to overcome the challenges of barriers transdermal technology, such as dense local tissue.
A key challenge of transdermal technology is the fabrication of a robust delivery platform that can work autonomously, that presents long-lasting operation and that is fully biocompatible. Microneedles according to the currently disclosed technology can enhance the release kinetics of antibody and any desired payload compared to actual methods and can easily pierce the skin due to its sharp tip. Furthermore, this currently disclosed technology offers multiple flexibilities regarding operation, dosage, storage, particle tuning size and release enhancing percentage.
Disclosed are devices, systems and methods for in-situ delivery of a cargo payload into tissues, human or non-human (e.g., animals, plants), via microneedles, including a degradable microneedle loaded with the payload that controllably releases the payload when inserted within the tissue. The microneedles according to some embodiments of the disclosed technology are loaded with the targeted payload along with active particles, which can serve as an active pump, and can enhance payload uptake and delivery in the organism.
Various examples of embodiments and implementations are disclosed. For example, in some implementations, the particle localized convective fluid transport of the presently disclosed technology remains active for more than 20 min, and the stability of these without application can last for long periods of time. The disclosed technology presents vast flexibility in terms of the size of the particle, pH environment and the concentration of the payload, is not limited to specific dimensions (diameter and height), a specific material (polymer), or active particles, and lastly, has been demonstrated to work in the delivery of therapeutic agents, antibodies, proteins, genetic material, particles, viruses, virus-like particles as well as in small or big therapeutic molecules.
Such active microneedle delivery can offer a faster and greater distribution of the target payload into the target tissue as compared to conventional delivery techniques. The ease of use of the disclosed transdermal technology can provide a fast, autonomous, fully biocompatible, and reproducible way of delivering payloads into deep tissue, and without the need of externally triggered equipment, e.g., as compared to diffusion-based methods and hypodermic standard needles. The disclosed technology provides a good alternative for treatment delivery that is not associated with pain, injury and, in some cases, extreme fear in patients. It has great potential in clinical translation due to its fully biocompatible and autonomous nature.
In some aspects, an autonomous, degradable and active microneedle delivery platform employs magnesium (Mg) microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The active microneedle patch uses the bodily fluids to activate these Mg particles, leading to an enhanced transdermal delivery of the loaded payload which could result in shorter times and better distribution when compared to passive microneedles. The embedded Mg particles can substantially increase the displacement of tracer microparticles through localized fluid convection resulting from their microbubbles production.
Example data and results of various implementations are described herein. For example, the drug release kinetics of example implementations of various embodiments were tested in vitro by measuring the amount of IgG antibody (Ab) (model drug) that passed through phantom tissue and pigskin barriers. Moreover, the active microneedle delivery mechanism was shown to induce an immune response in a syngeneic B16F10 mouse melanoma model with significantly longer life expectancy when compared to immune therapy anti-CTLA-4 or corresponding use of passive microneedles.
In some embodiments, a microneedle patch is configured for combinatorial delivery using spatially-resolved active and passive microneedle zones, toward fast and deep delivery along with slow sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of biomedical and personal applications in connection to improved delivery of different drugs, vaccines, cosmetics and gene therapy modalities, towards greatly improved outcome, convenience and costs.
In some embodiments, a microneedle patch is configured for virus immunotherapy delivery and distribution into a tumor tissue more effectively, therefore targeting the tumor microenvironment more broadly as compared to soluble bolus administration. The combination of active delivery and enhanced distribution greatly enhances efficacy of a plant virus cancer immunotherapy applied as in situ vaccine.
In some aspects, a microneedle therapeutic payload delivery device includes a substrate; an activation particle; and one or more degradable microneedle structures coupled to the substrate and including a polymeric matrix structured to embed the activation microparticle and one or more therapeutic payloads, the one or more degradable microneedle structures each including an exterior wall spanning outward from a base surface and forming an apex at a terminus point of the exterior wall, wherein the polymeric matrix of a microneedle structure of the one or more degradable microneedle structures is degradable under an environmental parameter of a biofluid surrounding the microneedle structure to dissolve and allow the one or more therapeutic payloads and the activation particle to the surrounding biofluid.
In some aspects, a method for autonomously delivering a payload into a biofluid via microneedles includes providing a microneedle patch device that includes a substrate and one or more degradable microneedle structures coupled to the substrate and including a polymeric matrix structured to embed a activation microparticle and one or more payload substances, the one or more degradable microneedle structures each including an exterior wall spanning outward from a base surface and forming an apex at a terminus point of the exterior wall, wherein the polymeric matrix of a microneedle structure of the one or more degradable microneedle structures is degradable under an environmental parameter of the biofluid that would surround the microneedle structure; applying the microneedle patch device to skin of a subject such that the one or more degradable microneedle structures are inserted into a tissue; dissolving the one or more degradable microneedles in the fluid based on exposure of the one or more degradable microneedles to the environmental parameter; and reacting the activation particle to the biofluid to cause the one or more payload substances to disperse away from the one or more degradable microneedle structures, thereby enhancing penetration of the one or more payload substances into the tissue.
The subject matter described in this patent document can be implemented in specific ways that provide one or more of the following features.
Current efforts and innovations on drug delivery platforms have the potential to enhance therapeutic efficiency. Existing therapy modalities (e.g., pill- or liquid-based oral delivery, needle-injections, suppository delivery, etc.) have successfully addressed basic medicine delivery requirements. Yet, there are urgent needs to develop local delivery platforms that can address and overcome the pain and fear from hypodermic injections and poor absorption and toxicity-related problems associated with the systemic delivery by pills-all while maintaining cost-efficacy and ease of use.
One plausible solution relies on the use of microneedles towards painless and localized delivery of drugs across the skin. For example, microneedles have proved useful for enhancing the drug permeability through the epidermis. Furthermore, this route offers autonomy and ease of use, as the therapeutic payload remains released over prolonged periods based on the material properties or by the inclusion of encapsulated smart drug-loaded particles.
Existing microneedle drug-delivery platforms usually rely on passive diffusion, which limits the penetration depth and distribution of the therapeutic payloads. While a gradual release of equivalent doses of therapeutics from transdermal microneedles can improve the efficacy compared to conventional treatment methods, diverse clinical circumstances may benefit from a rapid, burst payload release. For example, passive diffusion of drugs from the microneedles may still exhibit limited permeation within tumor microenvironments, where tissue penetration based solely on diffusion-based methods may also be more restricted. In this direction, diverse external stimuli have been employed to enhance the drug permeation through the epidermis; these external triggers include electroporation, ultrasound, light, and temperature, Yet, the requirement of external (often costly and bulky) equipment limits the widespread use of these stimuli to specialized centralized lab settings but not to field/remote locations. Thus, it is desirable to provide advantages of both autonomous and active delivery into a single platform, towards coupling of the benefits of autonomous delivery and reducing the time necessary for achieving high therapeutic efficiency.
Disclosed are devices, systems and methods for in-situ, active delivery of a payload into tissues via degradable microneedles. The disclosed devices, systems and methods utilize an environmental parameter of the biofluid to trigger degradation of one or more materials of the microneedles that encapsulate both a payload and an activation particle (e.g., microparticle or nanoparticle) to react in the biofluid and autonomously facilitate delivery of the payload to the surrounding biological environment. The disclosed devices, systems and method stand in contrast to convention microneedle techniques, which suffer from the limited diffusion (e.g., penetration depth) and inefficient distribution of the payload; and thus, require costly and bulky equipment.
In some aspects, disclosed is a degradable and autonomous active microneedle delivery platform based on a robust built-in micropump system, for enhanced payload permeation. For example, in some embodiments, a microneedle therapeutic payload delivery device includes a substrate; an activation particle; and one or more degradable microneedle structures coupled to the substrate and including a polymeric matrix structured to embed the activation microparticle and a therapeutic payload, the one or more degradable microneedle structures each including an exterior wall spanning outward from a base surface and forming an apex at a terminus point of the exterior wall, wherein the polymeric matrix of a microneedle structure of the one or more degradable microneedle structures is degradable under an environmental parameter of a biofluid surrounding the microneedle structure to dissolve and allow the therapeutic payload and the activation microparticle to the surrounding biofluid.
In some example embodiments, the microneedle patch includes a degradable polymeric microneedle body loaded with a therapeutic payload and active motor-Mg microparticles. In implementations, for example, once the microneedle patch is attached to a patient's skin and the polymeric microneedles are exposed to subcutaneous biofluid (e.g., interstitial fluid (ISF)), the polymeric microneedles are able to dissolve such that the embedded Mg particles react (e.g., instantaneously) with the surrounding the subcutaneous biofluid, resulting in a rapid generation of hydrogen bubbles, that induces distinct vortex flow fields, and lead to a powerful pumping-like action, and dynamic transport of the embedded therapeutic payload.
Several example embodiments and implementations of the disclosed degradable microneedle payload-delivery technology are described below. For example, as discussed in further detail later in this patent document, implementations of an example embodiment of the degradable microneedle patch examined the drug release kinetics (in an example model) to test in vitro the degradable and autonomous active microneedle delivery platform, e.g., by measuring the amount of therapeutic payload that passed through phantom tissue and pigskin barriers, which presented enhanced permeation and distribution when compared to passive microneedles. Moreover, the advantages of the active platform's therapeutic efficacy in an animal melanoma model are discussed herein.
The versatility of the example approach is demonstrated by integrating spatially-resolved active and passive microneedles in the same patch, which can provide combinatorial drug delivery including active and passive segments. Such active microneedle delivery offers ease of use as an “all in one device,” providing an autonomous, biocompatible, and efficient alternative for faster release kinetics of payloads through the skin while preventing the need of external activation (and related trigger equipment). Thus, it holds considerable promise to reduce the time necessary to achieve high therapeutic efficiency of different drugs, vaccines, and gene therapy modalities towards diverse biomedical applications.
In some aspects, the microneedle according to the presently disclosed technology can include a sharp array (solid or hollow) which serves to penetrate into the tissue. The microneedle material can be made from diverse transient degradable materials (e.g., polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), hyaluronic acid (HA), sodium alginate (SA), Pullulan, starches and/or sugars), that start to dissolve upon contact with physiological and low pH medium (e.g., less than pH 7). The use of the optional systems to monitor the delivery can be integrated into the needle.
The block diagram of
In various implementations of the active microneedle delivery system 100, the payload 102 is a therapeutic agent, which can be one or more of drugs, particles, molecules, genetic material, proteins, virus or viral vectors, and/or enzymes (or a combination thereof).
In various embodiments, the activation particle 103 can include a nanoparticle or a microparticle. For example, the activation particle 103 can be configured as a Mg microparticle that generates a propulsive force to drive the payload 102 into an affected area of the host to which the system 100 is deployed (e.g., a tumor).
In some embodiments of the active microneedle delivery system 100, for example, the substrate 104 includes an adhesive layer that adheres to the microneedle structure 101; and in some embodiments, the substrate 104 is a single adhesive material that binds to the base surfaces of the microneedle structure(s) 110. In some embodiments, the substrate 104 can include a medical adhesive (e.g., Tegaderm) and/or non-dissolving polymeric materials, e.g., polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), or other.
In some example implementations, the activation entity is an active microparticle comprised of Magnesium (Mg0) metal. Yet, notably, the disclosed microneedles can include but are not limited to the use of activation entities such as micro/nanoparticles (e.g., Mg0, calcium carbonate CaCo3, zinc Zn0), chemically modified micro/nanoparticles (micro/nanomotors) with biocatalytic enzymes (e.g., urease, catalase, glucose oxidase), spherical (Janus) and rocket type micro/nanomotors made of inorganic materials (e.g., Mg0, Zn0, gold Au0, titanium dioxide TiO2), or micro/nanoparticles modified metal organic frameworks (MOF) as catalytic engines and porous carriers (e.g., zeolitic imidazolate framework-67 and 8). These activation entities incorporated in microneedle patches include materials that enable the conversion of local chemical fuels (e.g., interstitial fluid, sweat), or external fields (e.g., magnetic, ultrasound, light) into leading forces (micropumps) within the application site, inducing fluid transport, enhancing and improving the distribution, penetration and efficacy of a therapeutic.
A wide variety of cargo payloads can be loaded and released, which include but are not limited to therapeutic agents, drugs, particles, molecules, genetic material, proteins, enzymes, etc., using singular or combination therapy. Diverse cargo therapeutics can be efficiently loaded within the microneedle embodiment, entrapped or coated in their free form or labeled/within micro/nanoparticles, which include but are not limited to: immune oncology agents, such as immune checkpoint inhibitors (e.g., anti-CTLA-4, anti-PD-1/PD-L1), chemotherapeutic agents (e.g., alkylating agents, mitotic inhibitors, antimetabolites, topoisomerase inhibitors), chronic pain medication (e.g., antidepressants, anticonvulsants, muscle relaxants, nonsteroidal anti-inflammatory drugs), cardiovascular medication (e.g., anticoagulants, ACE inhibitors, cholesterol-lowering agents, vasodilators), anti-aging agents (e.g., retinoids, ascorbic acid, hydroxy acids, antioxidants), antiviral therapeutics (e.g., non-retroviral antiviral agents, antiretroviral agents), vaccines (e.g., live attenuated, inactivated, recombinant, conjugated, toxoid), antibacterial drugs (e.g., inhibition of cell wall synthesis, inhibition of protein synthesis, inhibition of bacterial nucleic acid synthesis), micronutrients (e.g., vitamins, minerals) and gene editing effectors (e.g., CRISPR-Cas9, TALE nucleases), using singular or combinatorial microneedle patches for a fast or sustained delivery.
In some example implementations, the microneedles with active fluid transport can work as a pump towards enhancing mass transport and fluid mixing to increase the release kinetics and distribution of the payload, for example, to deliver insulin to a patient or towards oral delivery of anesthetic (e.g., like that illustrated in
In some example implementations, a schematic illustration of the active payload delivery microneedle patch 200 is illustrated in
The ability to load both active particles and a therapeutic payload in a single needle is illustrated by the fluorescence and digital microscopy photographs shown in
In some example implementations, active microneedles can be fabricated by a micromolding technique in accordance with the method 210 (
The active microneedles can react quickly after contacting fluids, as shown by the time-lapse microscopy images in
In some aspects, the presently disclosed technology has several advantages such as faster release kinetics, painless application, low cost, sharp tip, no sharp waste and easy of fabrication and practical use. The microneedle structure can have diverse dimensions and shapes, such as a 400 μm base and 850 μm height and have a sharp stiff tip similar to a bee sting (e.g., less than 50 μm). The microneedles can be filled with Mg particles, where polymer (e.g., PVP) casting and polymerization is subsequently carried out on top of the Mg particles, embedding the Mg particles in a polymer matrix. The outcome of this process allows the formation of shaped microneedle tips (
In some example implementations, the parameters relevant to the Mg active-delivery performance were examined to understand the active microneedles under different environments. The enhanced mixing of the active needle is dependent on the number of magnesium particles loaded. Based on findings that the peak value of random sphere packing of conical structures is 66.64%, it is estimated that a microneedle array that includes a total of 225 microneedle tips can load a total volume of ˜10.13 μL in the tips, thus providing flexible delivery possibilities. The theoretical volume loading capabilities of a single microneedle tip was calculated and plotted as shown in
In some example implementations, the close packing of the Mg particles within the microneedle is illustrated by an SEM of a single microneedle tip, filled with Mg particles, along with its corresponding EDX, corroborating the Mg successful inclusion within the microneedle structure (
The degradation rate of the Mg particles in the active and passive microneedles shows a slow degradation rate of Mg (
In some aspects, a combination of the presently disclosed transient polymer and active biodegradable particles upon its activation in biological media enhance the delivery of a payload. This technology can work for, but not limited for the enhanced delivery of antibody, protein, enzyme, and drug-small-large molecules. The microneedle dissolution rate changes significantly if active particle microneedles are compared to static and inert particles as well as only polymer microneedles. Mg active particles can pertain activity for more than 30 min in diverse pH environment, generating local fluid transport phenomena in the applied area thus, dissolving faster the microneedle and consequently a greater dispersion of the payload (
In some example implementations, prior to performing in vitro release kinetic experiments and further testing of the enhancing capabilities of active microneedles in payload delivery, the fluid mixing performance of the active Mg particles was evaluated in the presence of smaller tracer particles in solution. The polymer dissolution of different microneedle tips, by the variation of active particles, inert particles, or without any, varied as expected.
Time-lapse images of the dissolution rate in bare phosphate buffered solution pH 6 of different microneedles are presented as follows: polymer (e.g., PVP) (
In some example implementations, the embedded Mg particles were found to substantially increase the displacement of tracer microparticles through fluid convection resulting from their microbubbles production. A schematic of the fluid transport of Mg active particles in the presence of tracer particles is presented in
The Mean Square Displacement (MSD) of the tracer particle trajectories, in the absence and presence of the Mg particles 503, is shown in
The illustrative schematic of
In some example implementations, the accelerated and enhanced localized particle mixing was corroborated by a COMSOL Multiphysics simulation of the flow generated with and without particles in the microneedle structure.
−∇p+∇2u=ΣiFi
∇·u=0
where the summation is over the bubbles present in the fluid. As shown in
In Vitro Enhancing Payload Release from Microneedles
In some implementations of example embodiments of the active microneedle delivery system 100, the activation of the particles for enhancing the dissolution of the microneedle material (faster than normal) was investigated in vitro. It was studied in the example implementations, active microneedles loaded with a fluorescence-tagged antibody improved its distribution and permeation into phantom mimicking tissue when compared to actual diffusion methods (
In some example implementations, the in vitro payload release and the potential use of active particle microneedles was evaluated towards enhanced and accelerated therapeutic efficiency. The active microneedle release kinetics were evaluated by employing 3 different techniques: electrochemical, spectrophotometric, and fluorescence. Briefly, for the electrochemical measurements of the payload, active microneedles were loaded with a fixed concentration of 50 μg of a tagged IgG-HRP, this antibody was used as a payload model while its electrochemical detection was performed to compare the release kinetics of diverse approaches.
In some example implementations, the electrochemical set up used for the detection of the payload (e.g., antibodies) (represented in
A schematic of the microneedles including an antibody payload are further depicted and characterized in
In some example implementations, in
In some example implementations, an evaluation of the dye release performance of both passive and active microneedles was performed ex vivo onto porcine skin.
In some example implementations, the in vivo therapeutic efficacy of the active transport method was evaluated by treatment of syngeneic B16F10 mouse melanoma model under different conditions. Anti-CTLA-4 monoclonal blocking Ab was chosen as a model payload to treat melanoma, because it was one of the first immune checkpoint inhibitors tested and approved for cancer patients. Prior to treatment, B16F10 tumor cells were intradermally induced in the right flank of female C57BL/6 mice.
The immunotherapeutic efficacy of different treatment regimens was assessed by measuring the growth rate of the tumors over time as shown in
In the example implementation shown in
A scanning electron micrograph of the polymeric combinatorial microneedle patch is shown in
The combinatorial loading of both dyes in a single microneedle patch is further illustrated in
In some embodiments, microneedle patch is a combinatorial path comprising one or more therapeutic payloads. In some embodiments, the one or more therapeutic pay loads include a first therapeutic agent and a second therapeutic agent, wherein the microneedle patch is configured to release the first therapeutic agent in to a subcutaneous fluid prior to a release of the second therapeutic agent.
The solid tumor microenvironment (TME) poses a significant structural and biochemical barrier to immunotherapeutic agents. To address the limitations of tumor penetration and distribution, and to enhance antitumor efficacy of immunotherapeutics, provided herein is autonomous active microneedle (MN) system for the direct intratumoral (IT) delivery of a potent immunoadjuvant, cowpea mosaic virus nanoparticles (CPMV) in vivo.
In some embodiments, the active microneedle delivery system 100 includes magnesium (Mg) microparticles embedded into active microneedles that react with the interstitial fluid in the TME, generating a propulsive force to drive the nanoparticle payload into the tumor.
Example implementations are described showing active delivery of CPMV payload into B16F10 melanomas in vivo, which demonstrated substantially more pronounced tumor regression and prolonged survival of tumor-bearing mice compared to that of passive MNs and conventional needle injection. Active MN administration of CPMV also enhanced local innate and systemic adaptive antitumor immunity. This approach represents an elaboration of conventional CPMV in situ vaccination, highlighting substantial immune-mediated antitumor effects and improved therapeutic efficacy that can be achieved through an active and autonomous delivery system-mediated CPMV in situ vaccination.
Melanoma exhibits high levels of IT heterogeneity, with clonal populations arising due to a variety of selection pressures. For in situ vaccination, it is important to distribute CPMV evenly throughout the tumor tissue to attract adenomatous polyposis coli (APCs) to sample and present tumor antigens from all clonal populations within a single tumor. The MN patches described herein distribute the therapeutic payload over an array of MNs; and thus, offer more uniform delivery throughout the tumor volume. This reduces the reliance on the nonuniform techniques and abilities of administering physicians to disperse the therapeutic throughout the tumor. In addition, CPMV is a macromolecule (30 nm) than most immunotherapeutic agents that have been successfully administered via passive, diffusion-based MNs; thus, active delivery-based administration can enhance CPMV tissue delivery beyond that achieved with passive MNs.
Described herein are autonomous and biocompatible delivery platforms incorporating immunostimulatory CPMV nanoparticles and Mg-based active MN delivery into dissolvable biodegradable MN patches. Provided herein is the characterization of the active MN delivery patch, corresponding spatiotemporal distribution of the payload CPMV, the subsequent immune response, a demonstration of the enhanced therapeutic efficacy of such rapid CPMV release from active MNs for improved in situ vaccination against the B16F10 model of melanoma.
In some example implementations, the active MN delivery system is incorporated with a patch design to facilitate application in treatment of a murine dermal melanoma model. In designing the MN delivery system each of the following were factors considered in order to optimize the system: the materials of which the MNs as composed, the size of the MNs, and the size of the patch serving as the platform from which the MNs extend.
In some example implementations, the active MN patch is comprised of a water-soluble polymer matrix made of a high molecular weight Polyvinylpyrrolidone (PVP), which serves as an enclosure for the active Mg microparticles (e.g., 30-100 μm in diameter) and the therapeutic payload of CPMV nanoparticles, both loaded within the structure (
In some example implementations, the active MN patches comprising CPMV are fabricated by a micromolding process involving a negative polydimethylsiloxane (PDMS) MN molds as reusable templates (
In some example implementations, the microneedle system includes a circular patch with a thin polymeric base of ˜100 μm in thickness and ˜12 mm in diameter, attached to an array of microneedles (see
In some implementations, the MN microneedles including the CPMV dissolve upon contact with a fluid. To evaluate the release of CPMV nanoparticles from MNs in vitro, active MN patches were loaded with Cyanine3 dye (Cy3) conjugated-CPMV (Cy3-CPMV). The Cy3-CPMV was distributed within the MN structure and along the base (e.g., a thin polymeric film ˜100 μm in thickness). The Mg microparticles were confined and concentrated within each MN tip. Brightfield (e.g., Nikon Eclipse Instrument Inc. Ti-S/L100) and fluorescence microscopy images (e.g., EVOS FL microscope, RFP fluorescent filter) with immersion in phosphate buffered saline (e.g., PBS, pH 6.5) were then collected. Rapid dissolution of the active MN tips when immersed in solution with vigorous and spontaneous H2 bubble generation and Cy3-CPMV release was observed (see
In some implementations the active and passive MNs patches demonstrate a high degree of stability. For example, the active and passive MNs patches can be stable when maintained at room temperature and dry conditions for up to 2 months. The dry polymer PVP matrix can provide stability to both therapeutic payload and Mg microparticles without requiring refrigeration. In some implementations, the MN have a high degree of mechanical stability. Mechanical stability and strength requirements allow the MN to breach dermal barriers in vivo. Accordingly, an axial mechanical compression test on each MN was performed to evaluate its failure force. The mechanical strength results yielded a fracture force of 550 mN per MN tip (
CPMV In Situ Vaccination with Transdermal MN Patch Application for B16F10 Melanoma
In some example implementations, the active MN patches can be used for CPMV in situ vaccination. In other example implementations, the active MN patches can be used for intertumoral in B16F10 dermal melanoma (see
To facilitate patch placement on exophytic or irregular shaped larger tumors, the patches were cut to smaller pieces (e.g., 4-9 pieces) such that a full dose was administered over the contours of the mass with greater coverage of the tumor (see
In some implementations, the CPMV is potent when administered intratumorally via injection or MN patches. Greater tumor regression was observed in the passive and active MN-treated groups compared to injected CPMV-treated groups within the first three days post treatment. Injected CPMV did not lead to tumor regression in this time period, but rather appeared to slow progression relative to the PBS injection. Over the next 7 days, tumor progression was delayed the most in the active MN group, and progression was observed earlier in the CPMV and passive MN groups (see
In some example implementations, the active MNs exhibit an overall improvement in efficacy with a single treatment as compared to conventional techniques. When B16F10 melanomas reached approximately 25-30 mm3 in volume, 100 μg CPMV-loaded passive or active MN patches were applied to the tumors. Control mice were intratumorally injected with either PBS (30 μL) or CPMV (100 μg/30 μL PBS). The tumors were small and flat, permitting treatment without cutting the MN patches.
In some example implementations, after release from the active MNs, CPMV is widely distributed throughout the animal. Initial investigations involved determining that differences in distribution of CPMV released in vivo from active and passive MNs compared to IT injection may underlie differences in efficacy. Cy5-CPMV was employed to allow fluorescence imaging of CPMV nanoparticles (
Injection of CPMV resulted in high levels of fluorescence at the tumor site that gradually decayed over time.
For the active- and passive-MN treated tumors, the overall fluorescent signal detected was low and not significantly different from each other, nor the PBS group. The early tumor regression was still observed in these animals suggesting that the enclosed Cy5-CPMV was in fact released within these tumors. Some animals showed transient increase in signal in regions just inferior to the tumors, related to the tissue under the tumor into which the MNs were applied (
To further visualize the IT distribution of Cy5-CPMV, ex vivo immunofluorescence for employed of the in vivo treated tumors. B16F10 melanomas were generated and treated, as described above. Animals were euthanized and tumors resected en bloc and flash frozen in OCT at 24 h post treatment. Tumor sections were stained for blood vessels (CD31/PECAM-1), leukocytes (CD45), and cell nuclei (DAPI). Immunofluorescence of PBS treated tumor showed a vascular tumor with rare leukocytes. The Cy5-CPMV injection-treated tumor demonstrated uneven distribution of CPMV, with areas of greater Cy5-CPMV clustering seen as bright yellow regions at low power and high power (4× and 10× magnification, respectively). These brighter fluorescent areas of clustered Cy5-CPMV could be the appearance of the depots 24 h after injection. For the active and passive MN-treated tumors, Cy5-CPMV was observed to be less clustered within the tissues, bright yellow regions are not visible at low power. While at high power, discrete yellow puncta of Cy5-CPMV were observed, as compared to the larger, brighter clusters of Cy5-CPMV observed with conventional injection. Reduced clustering of Cy5-CPMV nanoparticles administered via MNs, would be consistent with the low fluorescence signal detected in the in vivo fluorescence microscopy images.
In some example implementations, the active MNs have an early cellular innate immune response. To investigate the early cellular innate immune response within the TME and its kinetics after treatment, the cellular immune infiltration of treated tumors was analyzed by flow cytometry at 4 and 24 h after either IT injection, passive MN, or active MN patch application (
As shown in
Examination of the subpopulations of CD45+ cells demonstrated distinctive changes in the IT CD45+ immune cell profile at 4 h and 24 h after treatment with different delivery systems (
The monocytic component (CD11b+ Ly6G−) of the CD45+ cell population exhibited complex, dynamic changes after the different methods of CPMV administration. The monocytic component included Type 1 tumor-associated macrophages (M1s, CD11b+F4/80+Ly6G−Ly6C−MHCII+CD86+), Type 2 tumor-associated macrophages (M2s, CD11b+F4/80+Ly6G−Ly6C−MHCII−CD86−), natural killer cells (NK cells, CD11b+NK1.1+Ly6G−Ly6C− F4/80−), monocytic-myeloid derived suppressive cells (M-MDSCs, CD11b+Ly6G−Ly6C+MHCII−SSClow), tumor-associated macrophages, whose phenotype was not further specified (TAMs not otherwise specified (NOS), CD11b+F4/80+Ly6G−Ly6C−MHCII+CD86− and MHCII−CD86+), and monocytic cells NOS (CD11b+Ly6G−). This NOS designation refers to the remainder of cells within a lower level gate that were not found in higher level gates with the flow cytometry gating strategy. For example, monocytic cells NOS refers to a population of cells that were found to be CD11b+Ly6G−, but did not fall into higher level gates where they would have been designated as M1, M2, TAMs NOS, NK, or M-MDSCs. TAMs NOS are the remainder of CD11b+F4/80+Ly6G−Ly6C− cells that were not also in the higher level MHCII+CD86+ or MHCII−CD86− gates, in which the cells were further specified as M1 or M2, respectively.
The immune response observed in active MN-treated tumors maintained a similar level of the monocytic component (CD11b+Ly6G−) of the CD45+ cell population relative to the PBS-treated tumors at 4 h post-treatment, while the levels in CPMV-injected and passive MN-treated tumors were relatively suppressed. Interestingly, while PBS-treated tumors and active MN-treated tumors had similar percentages of monocytic cells comprising the CD45+ population, within this component, the active MN-treated tumors had greater percentages of M1s and M-MDSCs than the PBS-treated tumors. The M1 percentage of active MN-treated tumors was also significantly greater than that of CPMV injection-treated or passive MN-treated tumors (
By the 24 h timepoint, the percentage of the monocytic component in PBS-injected tumors continued to increase. The monocytic component in the active MN-treated tumors decreased and CPMV injected-tumors increased until both reached comparable levels. The percentage in the passive MN-treated tumors increased to a similar level as that of the PBS-treated tumors (
The monocytic component consists of a mix of cells, including macrophages, monocytes, and M-MDSCs. Macrophages can exist in different functional states depending upon their surrounding environment. M1 macrophages promote a pro-inflammatory state and have antitumor activity. M2 macrophages promote tumor growth and progression. Several, sometimes conflicting, roles have been attributed to M-MDSCs. They are immunosuppressive and support tumor progression. However, there is evidence that they can also differentiate into different types of macrophages and DCs. The fate of these cells is at least partially influenced by the state of the surrounding TME. The early increase in M-MDSC percentage in the active MN-treated tumor could represent an increased pool of potential macrophages and DCs, and would be consistent with the increased percentage of active DCs and M1s, as well as M2s. Although at 4 h, the PBS-treated tumors had similar levels of M2s as the active MN-treated tumors, they also contained a reduced percentage of M-MDSCs, so these could have arisen from a different source or developmental pathway in the PBS-treated mice. Further, the monocytic cells NOS component represents a heterogeneous mix of monocytic cells, which include monocytes that also can differentiate into M1s or M2s. Macrophages have long life spans and enhanced phagocytic capacity, especially compared to that of neutrophils. Macrophages have been implicated as critical mediators of tumor regression through direct tumoricidal activity. The increased percentages of differentiated M1s, potential macrophage progenitors, and greater recovery of NK cells in active MN-treated tumors suggest a greater portion of the CD45+ infiltrate may be comprised of cells with pronounced ability to destroy tumor cells. This could underlie the augmented tumor regression observed in the tumors treated with active MNs. Passive MN-treated tumors appeared to have a delayed expansion of the monocytic cells NOS and M-MDSCs, but not M1 macrophages, at the 24 h timepoint. CPMV injection-treated tumors exhibited a more modest increase in its M1 percentage and NK cell recovery. Both CPMV injection and passive MN administration demonstrated inferior suppression of tumor growth.
With respect to granulocytic cell (CD11b+Ly6G+) and quiescent neutrophil (QN, CD45+CD11b−Ly6G+) components of the CD45+ infiltrate, dynamic changes in these subsets were also observed. The percentage of broadly granulocytic cells within CPMV injection-treated tumors expanded, compared to that of the PBS-treated tumors, and remained consistent at 4 h and 24 h after treatment (
TIN percentage did not differ from PBS-treated tumor levels in any groups at 4 h and 24 h after treatment (
The granulocytic cell percentage in active MN-treated tumors increased to a level comparable to that in the CPMV injection-treated tumors by 24 h, while the granulocytic component decreased in the passive MN-treated tumors (
It is believed that this increased efficacy and immune responses using the active MN-based CPMV administration is related to the differences in the kinetics of CPMV delivery. It further contemplated that increased efficacy is a result of the potential actions of the Mg micromotors themselves. Mg2+ is an important second messenger and has been implicated in T cell stimulation in response to antigens, macrophage development, M1/M2 polarization, and DC migration. These in vitro studies examined substantially higher extracellular Mg2+ concentrations (approximately 6 times greater) than the maximum possible concentration of Mg released from the active MN patch into the smallest volume (25 mm3) tumor in this study. While Mg2+ at higher concentrations plays an important role in T cell activation, it is unlikely that Mg2+ would reach distant lymphoid structures at concentrations relevant to CD8+ T cell activation in vivo. The relatively low Mg2+ concentration within the tumor and washout over time, would likely limit Mg2+ contribution to the overall enhancement of immune responses beyond small, short-lived effects. Moreover, in a previous report, active MN patches devoid of any payload therapeutic (blank MNs) did not demonstrate antitumor efficacy, as durable tumor growth suppression and overall survival in blank MN-treated mice did not differ significantly from those treated with PBS injection.
Overall, these results demonstrated that CPMV in situ vaccination via active MNs promoted enhanced IT recruitment and activation of APCs, including DCs and macrophages. The enhanced infiltration of macrophages is also consistent with the pronounced early tumor regression. Passive MNs, however, seemed to lack enhancement of activated DCs and M1s, with a delayed infiltration of other monocytic cells.
In some example implementations, the active MNs lead to an enhanced antitumor adaptive immune response. In situ vaccination optimally results in induction of a systemic antitumor response mediated by the adaptive immune system. Local innate immune activation is critical for priming these adaptive immune responses. To determine whether the remodeling of the TME and rapid infiltration of APCs by active MN treatment could effectively launch and improve the systemic antitumor response of CD8+ T cells, an interferon-γ (IFN-γ) release assay with splenocytes from treated mice was performed. CD8+ T cells producing IFN-γ indicates activation of the cells in response to recognition of their target antigen. B16F10 melanomas were treated when volumes reached 60 mm3. Splenocytes were isolated 10 days after treatment with PBS, CPMV injection, passive MN, or active MN as described above. The splenocytes were incubated with B16F10 melanoma cell lysate, CPMV, or culture media only in a suspension culture for 48 h. After this period, IFN-γ-producing effector CD8+ T cells (CD44hiIFN-γ+CD8+) frequency was evaluated using flow cytometry.
As shown in
Larger percentage of CD8+ T cells producing IFN-γ is associated with enhanced antigen priming and presentation by APCs, as well as greater suppression of tumor growth in other cancer vaccination strategies. CPMV treatment with active MNs promotes activation of a larger percentage of the CD8+ splenocyte population by CPMV or the targeted tumor. Increased APC cross presentation of CPMV antigens and tumor antigens with active MN treatment may mediate the heightened CD8+ T cell activation. Potential broader distribution of CPMV within the heterogenous tumor, with active MN administration, may also lead to APC collection and cross presentation of a more diverse array of tumor antigens in the lymphoid organs. In turn, this may induce activation of a broader subset of antitumor CD8+ T cells This finding supports the previous hypothesis that rapid, augmented APC infiltration into the TME with active MN-mediated CPMV delivery (
In some embodiments, MNs containing Mg microparticles can be used to deliver nanoparticles, specifically the plant viral nanoparticles, CPMV, for in situ vaccination against the B16F10 model of melanoma. In some embodiments, the MNs provide rapid release of CPMV from the active MN platform in vitro. Administration of CPMV with active MNs can enhance tumor regression compared to conventional injection.
In the example implementations described, fabrication of microneedles of different materials were performed. Microneedle arrays with a number of 225 tips had conical shape and presented dimensions of 1000 μm in height and 400 μm in base. Microneedles were made from polyvinylpyrrolidone with molecular weight of 360K. Microneedles were subjected to a variety of characterization methods, such as: imaging (Scanning Electron Microscopy), dissolution properties, and mechanical testing. Additionally, a micromolding process was performed to reproduce negative microneedle molds made from PDMS, and as well fabrication of phantom gel tissue samples.
In some example implementations, polyvinylpyrrolidone (PVP) average Mw˜360,000, PDMS base curing agent kit SYLGARD® 184, and agarose where purchased from Sigma Aldrich. HRP-Goat anti-human IgG Antibody (peroxidase) from Vector Laboratories. Goat anti mouse IgG-Alexa Fluor 555 from Abcam. Magnesium microparticles with size >=45 m, catalog #FMW40, from TangShanWeiHao Magnesium Powder Co., Ltd China. 0.9 μm Nile red fluorescent particles from Sphero Tech. Porcine skin was obtained from a near market. 3D printing UV sensitive resin was obtained from AnyCubic.
In some example implementations, the master microneedle mold was placed in a clean Petri Dish, Crystal Clear Borosillicate Glass with a double-sided tape to attach the mold properly. A mix ratio of 10:2 base/curing agent PDMS solution was later casted onto the microneedle patch and placed in vacuum within a desiccator for 5 min at 23 in of Hg. Bubbles were removed from the surface and PDMS was cured in an oven at 75 C for 30 min. Later sample was removed from the oven and cured PDMS was separated from petri dish gently to obtain the negative mold. PDMS mold was adjusted to desired size with the use of a blade cut.
Microneedle molds were washed with hand soap and rinsed with water twice, with further Ultrasonication bath for 15 min. Later, the mold was dried with air gun and cleaned by adding 0.25 mL of 2-propanol to each mold for 10 min. Molds were placed in the oven (75 C for 15 min) and not used until they reached room temperature.
In some example implementations, a volume of 0.25 mL of polymer (PVP) was added onto the previously cleaned PDMS microneedle mold and further placed in a closed desiccator in vacuum for 5-10 min (23 in Hg). Molds were removed from desiccator with the further removal of bubbles generated at interface between microneedle pores and solution with the use of plastic 1 mL disposable transfer pipettes or the use of a tweezer. Later, bubbles in the surface of the solution were removed, or popped with the use of a pipette or needle tip. Furthermore, a second addition of 0.25 mL of polymer was carried out, turning on vacuum again. This process was performed until polymer solution was 1 mL.
The payload solution (50 μg of IgG-HRP, 50 μg of IgG-AlexaFluor555, 20 μg of Rhd6G, 20 μg of FITC, or 100 ug of anti-CTLA-4) was added to the mold and let it to dry for 24-72 hours. Once microneedle patches were ready, a 1 cm2 scotch tape was applied on top of the needles and peeled off from the PDMS mold. Microneedle patches were stored at room temperature prior to use.
In some example implementations, 2% Agarose was weighted in a 20 mL Crystal Clear Borosilicate vial in DI water. Solution was heated a 175 C until solution turned transparent. Later, the temperature was lowered to 120 C and casted onto 1.5 mm, 3.0 mm or 4.5 mm Eco-Flex negative molds. Solution was let it dry for 2 min and further removed form mold with help of tweezers. Phantom skin mimicking gel were soaked in PBS pH 7.4 prior use.
In some example implementations, microneedle patches were characterized by Scanning Electron Microscopy in a FEI Quanta SEM System. The array was previously sputtered with an Iridium coating and placed on Quanta chamber to make it conductive. Microneedles were imaged at 2-5 KV spot n=3.
In some example implementations, a mechanical test was performed to polyvinylpyrrolidone microneedles by applying a constant load to a single tip. Microneedles show a force load capability (0.5 N/needle) by being good candidates for skin penetration. The mechanical strength of microneedles was measured by visualizing the displacement of the microneedle tip structure compared to the relative height of the cone vs the applied force with the use of a Force Gauge Model M4-20 system Mark0-10 Series 4.
In some example implementations, microneedles were subjected to a test of piercing and further dissolved into a 3 mm thickness 2% Phantom Tissue Skin with a PBS pH 7.4 reservoir below. Both passive and active microneedles (n=5) were loaded with 50 μg of Rhodamine 6G or IgG-HRP. After piercing for different set times, the supernatant was collected and analyzed by a Shimatzu UV-vis spectrophotometer from 300 to 700 nm. For the electrochemical measurement of IgG-HRP, a reservoir with TMB+H2O2 was placed below the phantom tissue and repetitive runs of amperometry at a fixed potential of +0.1V for 50 s were employed to analyze the current change behavior of both microneedle controls.
In some example implementations, a 1.5 mm thickness porcine skin area of 2×2 cm was pierced by microneedles for both diffusion and active control studies. The microneedle patches were placed for different interval times, 5, 10, and 20 min and further cross sectioned for analysis at room temperature.
In some example implementations, the B16F10 cell line was acquired from American Type Culture Collection (ATCC). B16F10 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies), supplemented with 10% (v/v) fetal bovine serum (FBS, Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin (Life Technologies). Cells were maintained at 37° C., 5% CO2. The cell cultures were maintained below a 50% confluence and early passage culture were utilized for the experiments.
In some example implementations, the described in vivo efficacy experiments were conducted in accordance with UCSD's Institutional Animal Care and Use Committee. 6- to 8-week-old female C57BL/6 mice (The Jackson Laboratory) were used. 25,000 B16F10 cells were suspended in 50 μL PBS and were injected intradermally into the right flank of C57BL/6 mice on day 0. PBS, anti-CTLA4 antibody (100 μg, Clone, 9H10, BioXcell) were administrated into mice by intratumoral injection (30 μL) or by microneedle on day 10 and day 17. Tumor volumes were measured using a digital caliper. The tumor volume (mm3) was calculated as (long diameter×short diameter2)/2. Animals were sacrificed when tumor volume reached 1500 mm3.
In some example implementations, the prototyping of solid microneedles is currently supported by paid or free commercial software (SolidWorks, Fusion360). The 3D microneedle STL models were transferred to a slicing software (AnyCubic Photon slicer64), which sliced the 3d model into thousands of micron layers in a 30M file, later connected to the printer via USB.
The file was uploaded to an AnyCubic Photon UV LCD 3D printer for the prototyping and printing. Microneedles were fabricated within a 115×65 mm build plate, by using exposure times of 8 s and step size of 20 um. This instrument projects a 25 W UV light source that sits inside a stainless-steel snoot, through a photocurable material. The 3D printer has a 2K LCD masking screen. 2560*1440(2 k) HD masking LCD provides very fine printing details down to few micrometers.
After the fabrication, the build plate containing microneedle devices was gently removed from the printer, and microneedle devices were detached. Supports printed to build the microneedle devices were removed, microneedles were rinsed in IPA and placed under an ultrasonic bath for the removal excess of uncured material. Microneedle devices were subsequently placed in a UV nail machine to post cure for 30 min.
In the example implementations described, fabrication of microneedles of different materials were performed. Microneedle arrays with a number of 225 tips had conical shape and presented dimensions of 1000 μm in height and 400 μm in base. Microneedles were made from polyvinylpyrrolidone with molecular weight of 360K. Microneedles were subjected to a variety of characterization methods, such as: imaging (Scanning Electron Microscopy), dissolution properties, and mechanical testing. Additionally, a micromolding process was performed to reproduce negative microneedle molds made from PDMS, and as well fabrication of phantom gel tissue samples.
In some example implementations, The fabrication of the PDMS negative MN molds was performed by casting a PDMS 8.6/1.4 (base/curing agent) solution (SYLGARD® 184) over a conical master MN mold made of acrylate resin (black-colored AnyCubic photon). Subsequently, PDMS was degassed for 15 minutes by placing the mold within a sealed desiccator connected to a vacuum pump running at 23 in Hg. Furthermore, the mold was left for 1 h at room temperature and later placed in an oven at 85° C. for 30 minutes. After the curing process, the negative mold was demolded from the master MN mold and resized with a blade cut. Prior use, each PDMS MN mold was cleaned/washed by triplicate with soap, ultrasonicated, temperature treated (80° C.), and stored in a sealed container.
In some example implementations, the active MN vaccination patches were fabricated by a micromolding technique with the use of negative PDMS MN molds. Briefly, 50 μL of a Mg microparticle (catalog #FMW40, from TangShanWeiHao Magnesium Powder Co., Ltd China) 2-propanol solution (50 mg/mL) was added to the negative MN mold to pack the cavities. Furthermore, a volume of 250 μL of a 10% w/v polyvinylpyrrolidone (PVP, MW=360 K, Sigma Aldrich) aqueous solution (pH 10.5 and pH 7.4) was casted over the negative molds in a closed desiccator at 23 in Hg for a total time of 10 minutes. Afterwards, bubbles were removed from the mold needle interface and repetitive additions of PVP solution were added to reach a total volume of 750 μL. The corresponding payload (100 μg of CPMV, Cy3-CPMV or Cy5-CPMV) was incorporated onto the mold and allowed to dry for 48 hours at room temperature in a sealed container. Upon drying, a circular 1.2 mm adhesive (3M scotch tape) was applied to the backing of the MN patches and demolded. Passive MNs were formulated by following identical preparation steps, however, the inclusion of Mg microparticles was not performed, respectively. Both active MN and passive MN patches were stored at room temperature for up to 2 weeks in a sealed container prior to use. For larger or more bulky tumors, MN patches were cut into 4 or 9 pieces to facilitate application.
In some example implementations, the fluorescent microscopy images of the active MN platform were performed by the use of an EVOS FL microscope (2× and 4× objectives and RFP fluorescent filter) for the Cy3-CPMV imaging. Furthermore, the SEM images were obtained with the use of a FEI Quanta 250 ESEM instrument (Hillsboro, Oreg., USA). Samples were sputtered with Iridium (Emitech K575X Sputter Coater) to provide a fine grain metal deposition and imaged with acceleration voltages between 3-5 keV. For the dissolution experiments, arrays of only 3 conical active MNs were attached horizontally to a clear glass slide. To capture the dissolution in real time, PBS pH 6.5 was added to the MN array and images were taken with the use of an inverted optical microscope (Nikon Eclipse Instrument Inc. Ti-S/L100) coupled with a 4× microscope objective, a Hamamatsu digital camera C11440, and a NIS Elements AR 3.2 software.
In some example implementations, after the MN patch fabrication, passive MN and active MN patches were used to pierce a phantom tissue. The synthetic phantom tissues were formulated with a 2% (w/v) Agarose (Sigma Aldrich) aqueous solution and further molded in custom made negative EcoFlex molds (1.5 mm diameter, 3 mm thickness). Phantom tissues were stored submerged in PBS (pH 6.5) and completely sealed prior to use. For testing, the passive MN and active MN patches loaded with Cy5-CPMV penetrated the phantom tissues for different durations: 1, 3, 5, 10, 20 and 30 minutes at 37.5° C. Following application, the patches were removed from the tissue and dissolved in 800 μL of PBS pH 6.5. The use of a UV-2450 Shimadzu spectrophotometer was used for the absorbance measurements from a 400-700 nm spectrum window and the release from patches was plotted vs time.
In some example implementations, the mechanical compression test was performed by the use of a Force Gauge Model M4-20 system Mark0-10 Series 4. In brief, an active MN array was set under a constant load, and the displacement of the base plate in reference to each needle height was monitored and plotted. The fracture (failure) force was determined by a notorious drop in force.
In some example implementations, the B16F10 cell line was acquired from American Type Culture Collection (ATCC). B16F10 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies), supplemented with 10% (v/v) fetal bovine serum (FBS, Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin (Life Technologies). Cells were maintained at 37° C., 5% C02. The cell cultures were maintained below a 50% confluence and early passage culture were utilized for the experiments.
In some example implementations, CPMV was propagated in California Blackeye No. 5 cowpea plants and purified. Bioconjugation of Cy3 and Cy5 fluorophores to CPMV external lysine residues. The CPMV protein capsid consists of 180 coat proteins upon which 300 surface-exposed lysine side chains are displayed.61 CPMV nanoparticles were labeled with sulfo-Cy5-NHS (Abcam) using N-hydroxysuccinimide-activated esters that target the surface lysine residues. The reactions were carried out with a 1,200-fold CPMV molar excess of sulfo-Cy5-NHS in a 0.1 M KP buffer (pH 7.0) at room temperature overnight, with agitation. This yielded approximately 30 Cy5 fluorophores conjugated to each CPMV. For Cy3 the reactions were carried out with a 3,000-fold CPMV molar excess of sulfo-Cy3-NHS, which yielded approximately 50-70 Cy3 fluorophores per CPMV. Fluorophore-conjugated CPMV was characterized by UV-visual spectral analysis, transmission electron microscopy, gel (SDS-PAGE and 1.2% (w/v) agarose) analysis.
In some example implementations, experiments were conducted in accordance with UCSD's Institutional Animal Care and Use Committee. Six- to eight-week-old female C57BL/6 mice (The Jackson Laboratory) were used. For larger tumors, 250,000 B16F10 cells were suspended in 30 μL PBS and were injected intradermally into the right flank of each C57BL/6 mouse on day 0. PBS (30 μL) or CPMV (100 μg in 30 μL) were administered by IT injection into the base of the tumor or by MN patch on day 7. MNs were applied on the tumors for 5-10 minutes until the needles completely dissolved. A PBS solution of pH 5.1 was applied to the skin of the treated region, immediately following application of the active MN patch. For smaller tumors, 25,000 B16F10 cells were suspended in 50 μL PBS and were injected intradermally into the right flank of each C57BL/6 mouse on day 0. PBS (30 μL) or CPMV (100 μg in 30 μL) were administered into mice by IT injection or by microneedle on day 10. Tumor volumes were measured using a digital caliper. The tumor volume (mm3) was calculated as (long diameter×short diameter2)/2. Animals were sacrificed when tumor volume reached ≥1500 mm3.
In some example implementations, 250,000 B16F10 cells were injected intradermally into the left flank of each C57BL/6 mice on day 0 as described previously. When tumors reached a volume of 60-100 mm3, IT PBS (30 μL) injection, Cy5-CPMV (100 μg in 30 μL) injection, passive MN, or active MN patch Cy5-CPMV (100 μg) were administered. 24 h post treatment, tumors excised en bloc from flank with 2-3 mm margin of normal surrounding skin. Tissue was flash frozen in OCT media with isopentane (cooled by dry ice to −78.5° C.). Tumors were cryo-sectioned into 5 m transverse sections (orthogonal to the longest axis). Tumor sections were fixed with cooled 100% acetone (−20° C.), then washed with PBS and blocked (1×PBS/5% (v/v) normal goat serum (Cell Signaling Technology, 5425S)/0.3% (v/v) Triton X-100) for 1 h at room temperature. Primary antibody staining was subsequently performed overnight at 4° C. with rabbit anti-mouse CD31/PECAMI polyclonal antibody (Abcam, ab28364) at 1:50 dilution and rat anti-mouse CD45 (Cell Signaling Technology, clone 30-F11) at 1:800 dilution. Sections were then washed in PBS and stained with secondary antibodies (anti-rabbit Alexa Fluor 568 (Abcam, ab175471) at 1:1000 and anti-rat Alexa Fluor 488 (Cell Signaling Technology, 4416S) at 1:500) at room temperature for 2 h. After washing with PBS and drying, sections were counterstained and cover slipped with Prolong® Gold Antifade Reagent with DAPI (Cell Signaling Technology, 8961S). Sections were visualized on Keyence BZ-X710 all-in-one microscope (Keyence Corporation) with filter set (DAPI, TRITC, FITC, and Cy-5) and accompanying imaging analysis software.
In some example implementations, the IVIS in vivo imaging system (IVIS Xenogen 200, Perkin Elmer) was used for non-invasive visualization and analysis of cutaneous distribution and retention of Cy5-CPMV administered via IT conventional injection, passive MN, and active MN in vivo. Mice were fed an alfalfa-free diet (PicoLab High Energy Mouse Diet, 5LJ5) starting at least lweek prior to imaging. 250,000 B16F10 cells were injected intradermally into the left flank of each C57BL/6 mouse on day 0 as described previously. When tumors reached a volume of 60-100 mm3, IT PBS injection, Cy5-CPMV injection, passive MN, or active MN patch Cy5-CPMV were administered, with doses as described previously. Mice were imaged with the Cy5.5 filter (excitation range 615-665 nm and emission range 695-770 nm, 0.5 s exposure) under anesthesia, before treatment (baseline or ‘BL’), and after treatment at specified timepoints (0 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h). Living Image Software (version 4.3.1, Perkin Elmer) was used to analyze all fluorescence data in this study. A region of interest (ROI) was drawn around each tumor and the measured fluorescence (in radiant efficiency, (p/sec/cm2/sr)/(μW/cm2)) was calculated and normalized to the baseline ROI fluorescence for each tumor.
In some example implementations, for tumor immunoprofiling and splenocyte interferon gamma (IFN-γ) release assays, fresh, single-cell suspensions were made from excised B16F10 melanomas and spleens, respectively. Cells were washed in cold PBS containing 1 mM EDTA, and then resuspended in staining buffer (PBS containing 2% (v/v) FBS, 1 mM EDTA, 0.1% (w/v) sodium azide). Fc receptors were blocked using anti-mouse CD16/CD32 (BioLegend) for 15 min and then tested with the following fluorescence-labeled antibodies (BioLegend) for 30 min at 4° C.: CD45 (30-F11), CD11b (M1/70), CD86 (GL-1), major histocompatibility complex class II (MHCII, M5/114.15.2), Ly6G (1A8), CD11c (N418 A), F4/80 (BM8), Ly6C (HK1.4), NK1.1 (PK136), CD4 (GK1.5), CD3ε (145-2V11 A), CD8α (53-6.7), CD44 (IM7), CD62L (MEL-14), and isotype controls. The gating strategy used for tumor immunoprofiling analyses were as previously described. The gating strategy used for splenocyte IFN-γ release assays are presented in
In conclusion, disclosed is an effective microneedle delivery route that offers active payload delivery, without the use of external stimuli, towards improved outcome vs commonly used passive diffusive microneedle transport. Such active degradable and autonomous microneedle delivery has been realized through the incorporation of Mg microparticles loaded within the microneedle patch, that produce a built-in pump source for faster and deeper intradermal local delivery. Such autonomous pumping action obviates the need for expensive and bulky external device essential for generating external stimuli. The presently disclosed technology allows the fabrication of a microneedle patch as well for combinatorial delivery using spatially-resolved active and passive microneedle zones, for fast/deep and slow sustained release, respectively. Moreover, the new active microneedle delivery system is not limited to specific polymeric materials or microneedle geometry or dimensions. The new “built-in” active delivery strategy holds considerable promise for diverse practical biomedical applications for transdermal delivery, including drug delivery, immunotherapy, along with cosmetic treatment, offering an attractive alternative to patients in the clinic when compared to traditional over the counter medical devices. In addition, the new active/passive combination delivery patch offers tremendous promise for pain killing and cardiac treatment applications towards diverse future biomedical applications in centralized and decentralized settings.
The presently disclosed technology has tremendous commercial promises as it could be implemented for diverse practical biomedical applications for transdermal drug delivery, immunotherapy, pain killing, and more. More specifically, this technology can lead to translation to the clinic due to its fully biocompatibility and autonomous nature.
In some embodiments in accordance with the present technology (example A1), an autonomous cargo delivery device comprising a microneedle patch that comprises one or more microneedles loaded with a therapeutic payload and an active microparticle.
Example A2 includes the device of any of examples A1-A5, wherein the one or more microneedles are degradable.
Example A3 includes the device of any of examples A1-A5, wherein the active microparticle reacts with subcutaneous biofluid upon insertion of the microneedle patch to enhance transport of the therapeutic payload.
Example A4 includes the device of any of examples A1-A5, wherein the active microparticle includes magnesium particles.
Example A5 includes the device of any of examples A1-A4, wherein the microneedle patch further comprises one or more passive microneedles.
In some embodiments in accordance with the present technology (example A6), a method of autonomous cargo delivery into a subject, comprising using a microneedle patch that comprises one or more microneedles loaded with a therapeutic payload and an active microparticle.
Example A7 includes the method of any of examples A6-A10, wherein the one or more microneedles are degradable.
Example A8 includes the method of any of examples A6-A10, wherein the active microparticle reacts with subcutaneous biofluid upon insertion of the microneedle patch to enhance transport of the therapeutic payload.
Example A9 includes the method of any of examples A6-A10, wherein the active microparticle is magnesium particles.
Example A10 includes the method of any of examples A6-A9, wherein the microneedle patch further comprises one or more passive microneedles.
In some embodiments in accordance with the present technology (example A11), a device for autonomous delivery of a molecular payload in tissue includes a substrate; and an array of microneedles on the substrate, the microneedles including a degradable polymeric microneedle body loaded with the molecular payload and a plurality of particles configured to react with surrounding subcutaneous biofluid when the microneedles are inserted in the tissue.
Example A12 includes the device of any of examples A11-A20, wherein the plurality of particles include magnesium microparticles capable to react with the surrounding subcutaneous biofluid to generate hydrogen bubbles that induce a vortex flow field to cause a dynamic transport of the molecular payload in the tissue.
Example A13 includes the device of any of examples A11-A20, wherein the dynamic transport of the molecular payload includes a pump-like action between the microneedles and the tissue.
Example A14 includes the device of any of examples A11-A20, wherein the plurality of particles includes a biocatalytic enzyme, an inorganic material, or a composite material configured to convert a local chemical fuel in the tissue or an external field applied at the tissue into leading forces that induce fluid transport.
Example A15 includes the device of any of examples A11-A20, wherein the biocatalytic enzyme includes glucose oxidase.
Example A16 includes the device of any of examples A11-A20, wherein the inorganic material includes a metal catalyst.
Example A17 includes the device of any of examples A11-A20, wherein the composite material includes a metal-organic framework composite.
Example A18 includes the device of any of examples A11-A20, wherein the external field includes a magnetic field, an ultrasonic field, or an optical field.
Example A19 includes the device of any of examples A11-A20, wherein the substrate includes a flexible substrate able to attach and conform to skin tissue.
Example A20 includes the device of any of examples A11-A19, wherein the device is configured to implement the method of any of examples A6-A10.
In some embodiments in accordance with the present technology (example B1), a microneedle therapeutic payload delivery device includes a substrate; an activation particle; and one or more degradable microneedle structures coupled to the substrate and including a polymeric matrix structured to embed the activation microparticle and one or more therapeutic payloads, the one or more degradable microneedle structures each including an exterior wall spanning outward from a base surface and forming an apex at a terminus point of the exterior wall, wherein the polymeric matrix of a microneedle structure of the one or more degradable microneedle structures is degradable under an environmental parameter of a biofluid surrounding the microneedle structure to dissolve and allow the one or more therapeutic payloads and the activation particle to the surrounding biofluid.
Example B2 includes the device of any of examples B1-B15, wherein the activation particle is configured to react with biofluid to enhance transport of the one or more therapeutic payloads into a tissue by dispersion of the one or more therapeutic payloads away from the one or more degradable microneedle structures and penetration of the one or more therapeutic payloads deeper into the tissue.
Example B3 includes the device of any of examples B1-B15, wherein the activation particle is microparticle or nanoparticle.
Example B4 includes the device of any of examples B1-B15, wherein the activation particle includes magnesium microparticles.
Example B5 includes the device of any of examples B1-B15, wherein the activation particle is coated with an enteric polymer.
Example B6 includes the device of any of examples B1-B15, wherein the activation particle is a chemically modified microparticle or nanoparticle with at least one of biocatalytic enzymes, an inorganic material, or a microparticle or nanoparticle modified metal organic frameworks (MOF).
Example B7 includes the device of any of examples B1-B15, wherein the polymeric matrix is formed of a transient degradable material including one or more of polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), hyaluronic acid (HA), sodium alginate (SA), or Pullulan.
Example B8 includes the device of any of examples B1-B15, further comprising one or more passive microneedle structures coupled to the substrate and including a nondegradable material, the one or more passive microneedle structures each including an external wall spanning outward from a base surface and forming an apex at a terminus point of the external wall.
Example B9 includes the device of any of examples B1-B15, wherein: the one or more therapeutic payloads includes a therapeutic agent selected from the group consisting of immune oncology agents, chemotherapeutic agents, chronic pain agents, cardiovascular agents, anti-aging agents, antiviral agents, vaccines, antibacterial agents, micronutrients, and gene editing effectors, and/or wherein the one or more therapeutic payloads includes a nanoparticle to which the therapeutic agent is attached; and/or the one or more therapeutic payloads includes a one or more of a drug, particle, molecule, genetic material, protein, virus-like particle, virus, enzyme, nanoparticle, or combination thereof.
Example B10 includes the device of any of examples B1-B15, wherein the one or more therapeutic payloads includes a first therapeutic agent embedded within a first microneedle structure of the one or more degradable microneedle structures, and a second therapeutic agent embedded within a second microneedle structure of the one or more degradable microneedle structures.
Example B11 includes the device of any of examples B1-B15, wherein the first therapeutic agent is releasable into the biofluid surrounding the microneedle structure before a release of the second therapeutic agent.
Example B12 includes the device of any of examples B1-B15, wherein the one or more therapeutic payloads includes a first therapeutic agent and a second embedded within at least one microneedle structure of the one or more degradable microneedle structures.
Example B13 includes the device of any of examples B1-B15, wherein the substrate includes an adhesive material on at least a side of the substrate interfaced with the one or more degradable microneedle structures.
Example B14 includes the device of any of examples B1-B15, wherein the environmental parameter includes a pH of less than 7.0.
Example B15 includes the device of any of examples B1-B14, wherein the biofluid includes interstitial fluid (ISF).
In some embodiments in accordance with the present technology (example B16), a method for autonomously delivering a payload into a biofluid via microneedles includes providing a microneedle patch device that includes a substrate and one or more degradable microneedle structures coupled to the substrate and including a polymeric matrix structured to embed a activation microparticle and one or more payload substances, the one or more degradable microneedle structures each including an exterior wall spanning outward from a base surface and forming an apex at a terminus point of the exterior wall, wherein the polymeric matrix of a microneedle structure of the one or more degradable microneedle structures is degradable under an environmental parameter of the biofluid that would surround the microneedle structure; applying the microneedle patch device to skin of a subject such that the one or more degradable microneedle structures are inserted into a tissue; dissolving the one or more degradable microneedles in the fluid based on exposure of the one or more degradable microneedles to the environmental parameter; and reacting the activation particle to the biofluid to cause the one or more payload substances to disperse away from the one or more degradable microneedle structures, thereby enhancing penetration of the one or more payload substances into the tissue.
Example B17 includes the method of any of examples B16-B27, wherein the activation particle includes magnesium, and wherein the reacting includes: oxidizing the magnesium of the activation particle from Mg0 to Mg+2 after reacting with H+ ions in the biofluid, resulting in production of gaseous H2, and generating a vortex within the biofluid by the gaseous H2, thereby mixing the one or more payload substances in the biofluid and driving the one or more payload substances deeper into the tissue.
Example B18 includes the method of any of examples B16-B27, wherein the activation particle includes an enteric coating.
Example B19 includes the method of any of examples B16-B27, wherein the activation particle is microparticle or nanoparticle.
Example B20 includes the method of any of examples B16-B27, wherein the activation particle is a chemically modified microparticle or nanoparticle with at least one of biocatalytic enzymes, an inorganic material, or a microparticle or nanoparticle modified metal organic frameworks (MOF).
Example B21 includes the method of any of examples B16-B27, wherein the polymeric matrix is formed of a transient degradable material including one or more of polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), hyaluronic acid (HA), sodium alginate (SA), or Pullulan.
Example B22 includes the method of any of examples B16-B27, wherein the tissue is a tumor, and the applying the microneedle patch device to the skin includes inserting the one or more degradable microneedle structures into the tissue without inserting the one or more degradable microneedle structures into one or both of blood vessels or underlaying subcutaneous tissue.
Example B23 includes the method of any of examples B16-B27, wherein the one or more therapeutic loads is an immune oncology agent and the enhanced transport of the one or more therapeutic loads induces an early cellular innate immune response in the biofluid.
Example B24 includes the method of any of examples B16-B27, wherein the tissue includes subcutaneous tissue.
Example B25 includes the method of any of examples B16-B27, wherein the one or more payload substances includes a therapeutic agent selected from the group consisting of immune oncology agents, chemotherapeutic agents, chronic pain agents, cardiovascular agents, anti-aging agents, antiviral agents, vaccines, antibacterial agents, micronutrients and gene editing effectors; and/or the one or more payload substances includes a one or more of a drug, particle, molecule, genetic material, protein, virus-like particle, virus, enzyme, nanoparticle, or combination thereof.
Example B26 includes the method of any of examples B16-B27, further comprising prior to the providing, attaching a therapeutic agent to a nanoparticle; and loading the nanoparticle with attached therapeutic agent into the polymeric matrix of the microneedle structure.
Example B27 includes the method of any of examples B16-B26, wherein the one or more payload substances includes a cowpea mosaic virus nanoparticle complex, the method comprising: applying the microneedle patch device to a region of the skin having a melanoma, such that the one or more degradable microneedle structures are inserted into the melanoma, wherein the reacting the activation particle cause the cowpea mosaic virus nanoparticle complex to disperse away from the one or more degradable microneedle structures via a propulsive force generated by the reacting to drive the cowpea mosaic virus nanoparticle complex into the melanoma to cause restructuring of a tumor microenvironment of the melanoma.
It is intended that the specification, together with the drawings, be considered exemplary only, where exemplary means an example. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of “or” is intended to include “and/or”, unless the context clearly indicates otherwise.
While this patent document contains many specifics, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this patent document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Moreover, the separation of various system components in the embodiments described in this patent document should not be understood as requiring such separation in all embodiments.
Only a few implementations and examples are described, and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.
This patent document claims priorities to and benefits of U.S. Provisional Patent Application No. 62/881,790 entitled “ACTIVE MICRONEEDLES FOR ENHANCED PAYLOAD UPTAKE” filed on Aug. 1, 2019. The entire content of the aforementioned patent application is incorporated by reference as part of the disclosure of this patent document.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/044660 | 7/31/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62881790 | Aug 2019 | US |
