Preparation and application of lipid nanogels for inhalation

Abstract

The application provides a liposome, which comprises a nanogel inner core and a phospholipid layer. The phospholipid layer is wrapped on the outer surface of the nanogel inner core, and the nanogel inner core comprises a degradable anionic polymer and metal ions. The degradable anionic polymer is connected with the metal ions through an ionic bond. The liposome prepared by the application overcomes the defects of traditional gel carriers, such as easy degradation in vivo, low delivery efficiency of macromolecular adjuvants, poor stability and high cost, and has the advantages of high in-vivo stability, high delivery efficiency, simple production process and low cost.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation and application of inhaled lipid nanogels, and more specifically, relating to liposomes, methods for preparing liposomes, pharmaceutical compositions, and the use of pharmaceutical compositions in the preparation of drugs. Background Technology

[0002] Lung cancer, classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), is one of the most common malignant tumors worldwide and the leading cause of cancer death globally, posing a serious threat to human health. Besides primary lung cancer, the lung is also one of the most common sites of tumor metastasis. Although studies have reported that radiotherapy and chemotherapy can improve patient survival rates, the cure rate remains low due to their cytotoxic effects and the development of drug resistance after multiple cycles of chemotherapy, hindering their clinical application. Currently, immunotherapy has achieved significant breakthroughs in clinical practice, providing new opportunities for treating various cancers, including lung cancer. Immune checkpoint inhibitors (such as anti-PD-L1 and anti-PD-1) have been shown to improve the survival rates of patients with melanoma, non-small cell lung cancer, and renal cell carcinoma. However, only a small percentage of patients are sensitive to checkpoint inhibitors. Further analysis indicates that low levels of anti-tumor T cells in patients are a significant reason for the failure of immune checkpoint inhibitor therapy; therefore, enhancing T cell responses is considered key to improving the efficacy of immunotherapy. Furthermore, due to the lack of specific tumor targets, only a small fraction of anti-PD-L1 antibodies effectively reach the tumor after systemic administration. Achieving therapeutic effects requires the use of large quantities of antibodies, increasing both toxicity and side effects, and leading to excessively high treatment costs. An ideal immunotherapy strategy should include effective immunotherapy, a sustained and specific anti-tumor immune response, and the use of low-dose drugs to reduce toxicity and treatment costs.

[0003] On the one hand, efficiently delivering immune agonists to antigen-presenting cells in the tumor microenvironment can enhance anti-tumor T-cell responses, thereby improving the efficacy of tumor immunotherapy. On the other hand, co-delivering immune checkpoint inhibitors to antigen-presenting cells in the tumor microenvironment can further relieve immunosuppression of T cells while reducing the toxicity of systemic administration. Given the unique advantages of non-invasive nebulized drug inhalation in the treatment of pulmonary diseases, delivering immune adjuvants and checkpoint inhibitors locally to pulmonary innate immune cells to activate anti-tumor T-cell responses is a promising treatment approach for lung cancer.

[0004] Polyinosinic-polycytidylic acid (poly I:C) is a double-stranded RNA that can act on multiple pattern recognition receptors (PRRs), such as TLR3, MDA-5, and RIG-I. These receptors primarily sense the invasion of RNA viruses and trigger corresponding T-cell immune responses. This multi-target mode of action can elicit a more robust immune response; for example, yellow fever virus vaccines can act on multiple PRRs, thus inducing a potent T-cell immune response in humans. However, poly I:C faces several barriers after inhalation. First, poly I:C is easily degraded by RNases in vivo, and the negatively charged nucleic acid molecules have difficulty effectively crossing the respiratory tract mucus layer to be phagocytosed by antigen-presenting cells. Second, after being phagocytosed by antigen-presenting cells, poly I:C is difficult to escape from the body and therefore cannot act on the cytoplasmic targets MDA-5 and RIG-I. In contrast, the MDA-5 / RIG-I targets induce higher levels of type I interferon-I secretion (IFN-I) compared to TLR3, thus better triggering T-cell immunity.

[0005] Therefore, there is an urgent need in the field to design a safe and effective delivery carrier that can simultaneously meet the above conditions, thereby promoting the translational application of polyICs as inhaled formulations in clinical practice. Summary of the Invention

[0006] This application is based on the inventor's discoveries and understanding of the following facts and problems:

[0007] While existing phosphatidylserine liposomes can facilitate the effective phagocytosis of cGAMP (Cyclic GMP-AMPP) small molecule adjuvants across the mucus layer by regulating the phospholipid components in the outer shell, their simple internal structure results in low efficiency of cGAMP reaching the cytoplasm after macrophage uptake, severely limiting their application. Therefore, the inventors designed a lipid nanogel formulation (LNG-pIC) by cross-linking bisphosphate-modified hyaluronic acid (HABP) and polyIC with calcium phosphate to form a nanogel core, which is then encapsulated with phospholipids. The resulting lipid nanogel formulation is less susceptible to nuclease degradation, exhibits improved stability of the nanogel core, increases the delivery efficiency of the large molecule adjuvant (polyIC), and is inexpensive, making it suitable for industrial production.

[0008] In a first aspect, the present invention provides a liposome. According to an embodiment of the present invention, the liposome comprises a nanogel core and a phospholipid layer; the phospholipid layer is wrapped around the outer surface of the nanogel core, and the nanogel core comprises a biodegradable anionic polymer and metal ions; the biodegradable anionic polymer and the metal ions are linked by ionic bonds.

[0009] Through experimental verification, the inventors discovered that the phospholipid layer on the surface of liposomes significantly enhances the physical stability of the nanogel core. Encapsulated by the phospholipid layer, the nanogel core can carry large-molecule nucleic acid adjuvant carriers without being degraded by nucleases in vivo. Simultaneously, it allows the formulation to effectively cross the mucus layer, while the internal nanogel core releases adjuvants within acidic endosomes. Through the proton sponge effect, the adjuvants further escape into the cytoplasm, stimulating corresponding signal transduction and type I interferon production, enhancing the anti-tumor T-cell immune response, and thus improving therapeutic efficacy.

[0010] According to embodiments of the present invention, the liposomes described above may further include at least one of the following additional technical features:

[0011] According to embodiments of the present invention, the biodegradable anionic polymer is selected from at least one of hyaluronic acid, hyaluronic acid modified with different bisphosphonates, or sodium alginate macromolecular polymers and their derivatives.

[0012] According to an embodiment of the present invention, the biodegradable anionic polymer is hyaluronic acid modified with bisphosphonate. According to an embodiment of the present invention, using hyaluronic acid modified with bisphosphonate can improve the cross-linking strength of the nanogel core.

[0013] According to an embodiment of the present invention, the metal ion is a calcium ion.

[0014] According to an embodiment of the present invention, the bisphosphonate-modified hyaluronic acid is linked to the calcium ion via a phosphate group or a carboxyl group.

[0015] According to an embodiment of the present invention, the mass ratio of the biodegradable anionic polymer to the metal ions is 0.45:2;

[0016] According to embodiments of the present invention, the phospholipid layer comprises at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids. The inventors have discovered that nanogels are extremely unstable in aqueous phases; however, by encapsulating the outer phospholipid shell using a microfluidic device, the overall stability is significantly improved. Furthermore, the phospholipid shell on the surface of the formulation enhances the physical stability of the HABP / PIC nanogel core while allowing the formulation to effectively cross the mucus layer and be phagocytosed by lung antigen-presenting cells (APCs), improving the adjuvant reflux effect and thus enhancing the antitumor immunotherapy efficacy of the formulation.

[0017] According to embodiments of the present invention, the phospholipid layer further includes cholesterol, PEG, or other auxiliary components.

[0018] According to an embodiment of the present invention, the nanogel core further comprises an adjuvant for activating T cells.

[0019] According to embodiments of the present invention, the adjuvant comprises any other component of a polyIC or TLR agonist.

[0020] According to embodiments of the present invention, any other components of the TLR agonist include CpG, R837, MPLA and their derivatives.

[0021] According to embodiments of the present invention, the nanogel core can carry 0.5%-10% adjuvant. Extensive experimental verification by the inventors has shown that the nanogel core carrying 0.5%-10% adjuvant can be expressed and function in organisms.

[0022] It should be noted that the minimum drug loading is 0.5% and the maximum drug loading is 10%.

[0023] According to an embodiment of the present invention, the optimal drug loading is 4.6%.

[0024] According to embodiments of the present invention, the adjuvant may be present in the form of being inserted into the phospholipid layer or being completely encapsulated within the nanogel core.

[0025] According to an embodiment of the present invention, the nanogel core further comprises siRNA, and the nanogel core may carry 0.5%-10% siRNA.

[0026] According to an embodiment of the present invention, the optimal amount of siRNA carried is 0.95%.

[0027] According to an embodiment of the present invention, the siRNA is used to interfere with the expression of immunosuppressive factors.

[0028] According to embodiments of the present invention, the siRNA includes at least one of PD-L1 siRNA, CTLA4 siRNA, and PD-1 siRNA.

[0029] According to embodiments of the present invention, the siRNA is selected from PD-L1 siRNA. According to embodiments of the present invention, local inhalation of PD-L1 siRNA can activate T cell responses while simultaneously relieving immunosuppression of T cells, thus improving therapeutic efficacy. Furthermore, local application of pIC and siRNA avoids the potential toxic side effects of traditional systemic activation or systemic use of checkpoint inhibitors, further improving safety.

[0030] In a second aspect, the present invention provides a method for preparing liposomes. According to an embodiment of the invention, the method includes: cross-linking a degradable anionic polymer in the presence of a cross-linking agent to obtain a nanogel core; and encapsulating the nanogel core in the presence of phospholipids to obtain the liposomes. According to an embodiment of the invention, compared to the high cost and stringent production processes of other T-cell therapies such as CAR-T and TCR-T, this lipid nanogel-based inhaled nucleic acid formulation is significantly effective, simple to process, and lower in cost, bringing hope to more patients.

[0031] According to an embodiment of the present invention, the degradable anionic polymer and an adjuvant undergo the crosslinking reaction in the presence of a crosslinking agent to obtain the nanogel core.

[0032] According to an embodiment of the present invention, the mass ratio of the biodegradable anionic polymer, crosslinking agent, and adjuvant is 0.2:3:0.1.

[0033] In a third aspect, the present invention provides a method for preparing liposomes. According to embodiments of the invention, the method includes: mixing a biodegradable anionic polymer, sodium dihydrogen phosphate, polyIC, and phospholipids using a microfluidic device to obtain a mixture of phospholipid-coated anionic polymer and polyIC; dialysis followed by incubation with calcium chloride (calcium ions can cross the phospholipid bilayer and form calcium phosphate with the inner phase sodium dihydrogen phosphate) to crosslink the anionic polymer to obtain the lipid nanogel. The inventors have found that the lipid nanogel prepared by microfluidic control encapsulates polyICs with an efficiency of up to 80%, effectively regulating the cytoplasmic delivery of the polyIC contents, inducing greater IFN-I secretion, and a more potent T-cell immune response. This formulation can be used for the efficient delivery of other nucleic acid molecules such as PD-L1 siRNA, further improving the efficacy of lung cancer treatment, and is inexpensive and suitable for industrial-scale preparation.

[0034] According to an embodiment of the present invention, the method further includes mixing the biodegradable anionic polymer, phospholipid, crosslinking agent and adjuvant to obtain the liposomes.

[0035] According to an embodiment of the present invention, the mixing process is performed in a microfluidic device.

[0036] According to an embodiment of the present invention, the mass ratio of the biodegradable anionic polymer, phospholipid, crosslinking agent, and adjuvant is 0.45:10:2:0.675.

[0037] According to an embodiment of the present invention, the crosslinking agent is calcium phosphate.

[0038] It should be noted that calcium chloride was actually added during the experiment, because calcium chloride reacts with sodium phosphate to form calcium phosphate after crossing the membrane. Therefore, it is calcium phosphate that actually performs the cross-linking effect.

[0039] In a fourth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the liposomes described in the first aspect of the present invention. The inventors have found that, upon administration, the pharmaceutical composition can stimulate corresponding signal transduction and type I interferon production in vivo, enhance anti-tumor T-cell immune responses, thereby improving therapeutic efficacy, and exhibits high safety.

[0040] According to embodiments of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0041] In a fifth aspect, the invention proposes the use of the liposomes described in the first aspect or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for treating or preventing disease. As previously described, the liposomes of the embodiments of the present invention can effectively cross the mucus layer, while the internal nanogel can release adjuvants in acidic endosomes, and further promote the escape of adjuvants into the cytoplasm through the proton sponge effect to stimulate corresponding signal transduction and type I interferon production, enhance anti-tumor T cell immune responses, thereby improving therapeutic efficacy, and have high safety.

[0042] According to an embodiment of the present invention, the disease includes cancer.

[0043] According to embodiments of the present invention, the cancer includes primary lung cancer, melanoma, breast cancer, and colon cancer.

[0044] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0045] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0046] Figure 1 The 1H NMR spectrum and characteristic peaks (H2) of HA-BP according to Example 1 of the present invention are shown. 1 -NMR) results graph;

[0047] Figure 2 This is a schematic diagram of the microemulsion synthesis of nanogels (NG-pIC) according to Example 1 of the present invention;

[0048] Figure 3This document describes the preparation and characterization of lipid nanogels according to Example 1 of the present invention. Figure A shows a schematic diagram of the lipid nanogels prepared by microfluidic method; the stability of the nanoparticles after incubation in BALF at room temperature for 2 hours or 24 hours was determined by measuring the hydrated particle size (B) and PDI (C); Figure D shows the zeta potentials of NG, LNP, and LNG; Figure E shows the encapsulation efficiency of polyICs in LNP and LNG as detected by agarose gel electrophoresis; Figure F shows the stability of bare NG and liposome-encapsulated NG after incubation in BALF at 37°C for 24 hours.

[0049] Figure 4 According to Embodiment 2 of the present invention, LNG-encapsulated polyICs were delivered to lung APCs via nebulized inhalation. Figure A shows representative in vitro fluorescence imaging of major tissues and organs of B16F10 lung-transfer mice at 1 h, 24 h, and 48 h after inhalation of DiR-labeled LNG-pICs; Figure B shows that the fluorescence signal was concentrated entirely in the lungs, and the fluorescence signal in the lungs was quantified (n = 5, *P < 0.05, ***P < 0.001); Figure C shows the quantification of fluorescence in various organs of B16F10 lung-transfer mice; Figure D shows the percentages of DiR+AMs, DiR+IMs, and DiR+DCs detected at 1 h, 24 h, 48 h, and 96 h after inhalation of DiR-labeled LNG-pICs; Figure E shows the percentages of DiR+mAMs, DiR+mCD103+DCs, and DiR+mCD11b+DCs that migrated to the TDLN at different time points after inhalation of DiR-labeled LNG-pICs, representing the total number of APCs.

[0050] Figure 5 According to Example 3 of the present invention, inhaled LNG-pIC can effectively stimulate the production of type I interferon in mouse lung tissue;

[0051] Figure 6 According to Example 4 of the present invention, inhaled LNG-pIC can effectively treat lung metastases from B16F10 melanoma. Using 2×10 5 A B16F10 melanoma lung metastasis model was established by intravenous injection of B16F10 cells into C57BL / B6 mice. On days 7 and 12 post-modeling, mice were treated with different preparations containing polyIC via inhalation. Figure A shows representative lung images (n=3) of mice sacrificed on day 17 (n=6 / group); Figure B shows statistical analysis of lung metastases (*P<0.05, ***P<0.001, ****P<0.0001).

[0052] Figure 7 According to Example 5 of the present invention, inhaled LNG-pIC inhibits the growth of B16F10 melanoma lung metastases by increasing immune cells. 2×105 B16F10 cells were intravenously injected into C57BL / B6 mice to establish a B16F10 melanoma lung metastasis model. On days 7, 12, and 17 post-modeling, mice were treated with different preparations containing polyIC via inhalation. Mice were sacrificed on day 18, and flow cytometry was used to analyze changes in CD80+CD86+, CD8+, and NK1.1+ T lymphocytes induced by different treatment groups. In this analysis, A and B represent the percentage of CD80+CD86+ T lymphocytes, C and D represent the percentage of CD8+ T lymphocytes, and E and F represent the percentage of NK1.1+ T lymphocytes (n = 7, *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0053] Figure 8 This is an example of inhaled LNG-pIC / siRNA combined therapy for B16F10 melanoma lung metastases according to Embodiment 6 of the present invention. 2×10 5 A B16F10 melanoma lung metastasis model was established by intravenously injecting B16F10 cells into C57BL / B6 mice. On days 7 and 12 post-modeling, mice were treated with different preparations containing polyIC via inhalation. Figure A shows representative lung images (n=3) of mice sacrificed on day 17 (n=5 / group); Figure B shows statistical analysis of lung metastases; Figure C shows representative lung images (n=3) of mice with B16F10 melanoma lung metastasis model who were intraperitoneally injected with anti-CD8 / anti-CD4 / anti-NK 1.1 and sacrificed on day 20; Figure D shows statistical analysis of lung metastases (*P<0.05, **P<0.01, ***P<0.001) ****P<0.0001). Detailed Implementation

[0054] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0055] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0056] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0057] In one aspect of the invention, a liposome is provided. According to an embodiment of the invention, the liposome comprises a nanogel core and a phospholipid layer; the phospholipid layer is wrapped around the outer surface of the nanogel core, and the nanogel core comprises a biodegradable anionic polymer and metal ions; the biodegradable anionic polymer and the metal ions are linked by ionic bonds.

[0058] According to embodiments of the present invention, the biodegradable anionic polymer is selected from at least one of hyaluronic acid, hyaluronic acid modified with different bisphosphonates, or sodium alginate macromolecular polymers and their derivatives, preferably hyaluronic acid modified with bisphosphonates.

[0059] According to an embodiment of the present invention, the metal ion is a calcium ion. In this paper, the inventors crosslinked bisphosphonic acid modified hyaluronic acid (HABP) and polyIC with calcium phosphate to form a nanogel core, and then encapsulated the core with phospholipids to prepare a lipid nanogel formulation (LNG-pIC). The calcium ion originates from the calcium phosphate in the crosslinking agent.

[0060] According to an embodiment of the present invention, the bisphosphonate-modified hyaluronic acid is linked to the calcium ion via a phosphate group or a carboxyl group.

[0061] According to an embodiment of the present invention, the phospholipid layer comprises at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids.

[0062] According to embodiments of the present invention, the phospholipid layer further includes cholesterol, PEG, or other auxiliary components.

[0063] According to embodiments of the present invention, a core-shell structured nanocarrier is developed by encapsulating an outer phospholipid shell using a microfluidic device for the safe and efficient delivery of macromolecular adjuvants. It offers the following advantages at the tissue, cellular, and intracellular levels: at the tissue / organ level, the nanolipid carrier can effectively cross the respiratory tract mucus layer, improving the lymph node return effect of the adjuvant; at the cellular level, the carrier can improve the efficiency of adjuvant uptake by antigen-presenting cells; and at the intracellular level, the carrier can facilitate adjuvant escape from endosomes, thereby enhancing adjuvant potency.

[0064] According to an embodiment of the present invention, the nanogel core further comprises an adjuvant for activating T cells.

[0065] In this article, the adjuvant refers to a non-specific immune enhancer that, when injected together with or pre-injected into the body with an antigen, can enhance the body's immune response to the antigen or alter the type of immune response.

[0066] According to embodiments of the present invention, the adjuvant comprises any other component of a polyIC or TLR agonist.

[0067] In this paper, the TLR (Toll-like receptor) agonist can activate antigen-presenting cells (APCs) and enhance T cell immunity against tumor neoantigens. Combined use with such innate immune agonists can improve the antitumor activity of immune checkpoint inhibitors. Furthermore, the vector constructed in this invention enables negatively charged nucleic acid adjuvants to effectively cross the respiratory tract mucus layer and be phagocytosed by pulmonary antigen-presenting cells (APCs), improving the adjuvant's lymph node reflux effect and thus enhancing its antitumor immunotherapeutic efficacy.

[0068] According to embodiments of the present invention, the adjuvant may be present in the form of being inserted into the phospholipid layer or being completely encapsulated within the nanogel core.

[0069] According to an embodiment of the present invention, the siRNA is used to interfere with the expression of immunosuppressive factors.

[0070] According to embodiments of the present invention, the siRNA includes at least one of PD-L1 siRNA, CTLA4 siRNA, and PD-1 siRNA.

[0071] According to an embodiment of the present invention, the siRNA is selected from PD-L1 siRNA.

[0072] In this paper, siRNA is a type of double-stranded RNA molecule, 20-25 base pairs in length, similar to miRNA, and operates within the RNA interference (RNAi) pathway, interfering with the post-transcriptional degradation of mRNA of specific genes expressing complementary nucleotide sequences, thereby preventing translation. The siRNA described in this invention is formed by the intracellular cleavage of double-stranded RNA (dsRNA) into 21-25 bp double-stranded RNA molecules using RNase III (such as Dicer). dsRNA can be exogenous, such as viral RNA replication intermediates or artificially introduced dsRNA; or endogenous, such as dsRNA formed from single-stranded RNA in cells under the action of RNA-dependent RNA polymerase. The inventors have discovered that local inhalation of PD-L1-loaded siRNA can activate T cell responses while simultaneously relieving T cell immunosuppression, thus improving therapeutic efficacy. Furthermore, local application of pIC and siRNA avoids the potential toxic side effects of traditional systemic activation or systemic use of checkpoint inhibitors, further improving safety.

[0073] In another aspect, the present invention provides two methods for preparing liposomes. According to embodiments of the present invention, the methods include a stepwise method and a one-step method.

[0074] The stepwise method involves cross-linking a degradable anionic polymer in the presence of a cross-linking agent to obtain a nanogel core. Alternatively, an adjuvant can be selectively loaded into the nanogel core, followed by encapsulation of the nanogel core in the presence of phospholipids to obtain the liposomes. According to embodiments of the present invention, compared to the high cost and stringent production processes of other T-cell therapies such as CAR-T and TCR-T, this lipid nanogel-based inhaled nucleic acid formulation offers significant efficacy, simple processing, and lower cost, bringing hope to more patients.

[0075] The one-step method involves mixing a biodegradable anionic polymer, phospholipids, and a cross-linking agent, with the option of selectively adding an adjuvant. The reaction is then carried out using a microfluidic device to obtain the liposomes. According to embodiments of the present invention, the lipid nanogels prepared via microfluidic control achieve an encapsulation efficiency of up to 80%, effectively regulating the cytoplasmic delivery of the polyIC contents, inducing increased IFN-I secretion, and a more potent T-cell immune response. This formulation can be used for the efficient delivery of other nucleic acid molecules such as PD-L1 siRNA, further improving the efficacy of lung cancer treatment, and is cost-effective for industrial production.

[0076] According to an embodiment of the present invention, the crosslinking agent is calcium phosphate. Calcium phosphate is a natural biomaterial with good biocompatibility and biodegradability. During the precipitation of calcium phosphate, excess calcium ions on the surface of the nanoparticles bind to anionic polymers. Intracellularly, acid-sensitive calcium phosphate degrades within the endosomes and releases the anionic polymers, while simultaneously altering the permeability of the endosomes, further releasing them into the cytoplasm.

[0077] In this paper, the cross-linking agent refers to a class of small molecule compounds, generally with a molecular weight between 200 and 600, possessing two or more reactive ends targeting specific groups (amino, thiol, etc.), which can couple with two or more molecules individually to bind them together. The cross-linking agent can covalently cross-link under physiological conditions, and the cross-linking reaction is rapid and simple. A variety of reactive groups are available, and the reaction can be either specific or non-specific. Furthermore, the cross-linking agent contains intercalary arms of varying lengths, effectively reducing steric hindrance. Some products incorporate cleavable groups, greatly enhancing the flexibility of cross-linking agent applications. The cross-linking agents described in this paper can be applied to: cell membrane structure research, protein structure research, protein-protein interaction research, biological missile research, the conjugation of carrier proteins and haptens, the immobilization of proteins or other molecules, antibody labeling, label transfer, and the conjugation of proteins and nucleic acids.

[0078] In another aspect of the invention, a pharmaceutical composition is provided. According to embodiments of the invention, the pharmaceutical composition comprises the liposomes described in the first aspect of the invention.

[0079] According to embodiments of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0080] In this document, the term "pharmaceutically acceptable" indicates that a pharmaceutical composition can be administered to a subject without producing adverse physiological reactions that would impede the administration of the pharmaceutical composition. For example, "pharmaceuticalally acceptable excipients" refers to excipients useful in the preparation of generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of such excipients or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous mediators, such as non-volatile oils, may also be used.

[0081] In this document, the term "pharmaceutically acceptable carrier" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples may include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to extend the shelf life or potency of antibodies.

[0082] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0083] The liposomes of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquids, nebulizers, semi-solid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The liposomes can be administered by nebulization, intravenous infusion, intramuscular injection, or subcutaneous injection.

[0084] The liposomes of the present invention can be inhaled into the lungs via atomization, taken up by antigen-presenting cells in the lungs, and then returned to the lymph nodes to efficiently activate the body's immune system.

[0085] In another aspect of the invention, the invention provides for the use of the aforementioned liposomes or pharmaceutical compositions in the preparation of a medicament for the treatment or prevention of disease.

[0086] According to embodiments of the present invention, the disease includes cancer. In this document, the cancer includes primary lung cancer, melanoma, breast cancer, colon cancer, and any cancer capable of metastasizing to the lungs.

[0087] In this article, the liposome or pharmaceutical composition can be used for the treatment of tumors, or in combination with other therapies such as immune checkpoint inhibitors such as PD-L1 siRNA to enhance the body's immune response, avoid the side effects of systemic use of checkpoint inhibitors, or be used for the prevention and treatment of infectious diseases.

[0088] In this paper, the microemulsion method refers to the formation of an emulsion by two immiscible solvents under the action of a surfactant. Nanoparticles are then obtained through nucleation, aggregation, and heat treatment within microbubbles. Its characteristics include monodispersity and good interfacial properties, and it is widely used to prepare group II-VI semiconductor nanoparticles. Microemulsions are thermodynamically stable, transparent monodisperse systems formed by water droplets in oil (w / o) or oil droplets in water (O / W). Their microstructures have particle sizes of 5–70 nm and are classified into O / W type and w / o (reverse micelle) type, representing ordered assemblages of surfactant molecules at the oil / water interface. As described in this paper, liposomes are prepared using the microemulsion method. The "microemulsion" is a clear, transparent, isotropic, and thermodynamically stable system composed of water, oil, surfactant, and co-surfactant. In microemulsion systems, water-in-oil (W / O) microemulsions are generally used to prepare nanoparticles. In W / O microemulsions, the water cores are surrounded by a monomolecular interfacial layer composed of surfactants and co-surfactants, dispersed in the oil phase. These water cores solubilize a certain concentration of reactants. Two microemulsions, A and B (A and B mixed can form a nanogel), are prepared, each containing reactants A and B respectively. Due to collisions, fusion, separation, and recombination between the micelles, the exchange or transfer of substances within the water cores occurs, causing a chemical reaction within the core, and the product (i.e., the nanogel) is formed within the water core. It should be noted that the liposome preparation method described above is mainly used to prepare the nanogel core, i.e., the control group in the examples.

[0089] The embodiments will be described in detail below. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0090] In this paper, EDC represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, a water-soluble carbodiimide used as an activator of carboxyl groups in amide synthesis, as well as for activating phosphate groups, cross-linking proteins and nucleic acids, and preparing immunoconjugates. The EDC used in the embodiments of this invention was purchased from Beijing Bailingwei Technology Co., Ltd., product number: 25952-53-8.

[0091] In this document, NHS (N-hydroxysuccinimide) refers to an uncharged SuLfo-NHS (N-hydroxysulfosuccinimide) analogue that can control and modify carbodiimide crosslinking reactions involving carboxylate (-COOH) activated by coupling with a primary amine (-NH2). The NHS used in the embodiments of this invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: 6066-82-6.

[0092] Example 1: Preparation of lipid nanogels

[0093] 1.1 Synthesis and Characterization of Bisphosphonic Acid-Modified Hyaluronic Acid

[0094] Bisphosphonic acid (BP) is coupled to the hyaluronic acid backbone via an amidation reaction between the -NH2 group on bisphosphonic acid and the -COOH group on hyaluronic acid (HA). According to an embodiment of the present invention, catalysis is carried out in an aqueous phase using an amidation catalyst, followed by dialysis to remove unreacted raw materials such as bisphosphonic acid and the catalyst. Specific steps are as follows:

[0095] 1) Dissolve 1 equivalent (eq) of HA in water to prepare a 10 mg / mL HA solution, and then add 2 eq of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide).

[0096] 2) Adjust the pH to 4-5 using NaOH or HCl, and react for 45 minutes. After 45 minutes of reaction, adjust the pH to 6-7 using NaOH or HCl.

[0097] 3) Add 2.5 eq of sodium alendronate to the reaction solution in step 2), maintain the pH at 6-7 and react for 2 hours, then react at room temperature for 3 days. After 3 days, dialyze the solution in ultrapure water for 3 days using a dialysis bag with a permeability of 8000-14000 molecular weight to remove unreacted small molecule raw materials.

[0098] The results showed that the substitution efficiency was estimated to be 10%-70% based on the characteristic peaks of BP replacing HA detected by 1H-NMR at 1.5-1.8 ppm. Figure 1 Other amidation catalysts include DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride), DMAP (4-dimethylaminopyridine), HOBT (1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), and TBTU (O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboronic acid). Characteristic peaks of 1.5-1.8 ppm after BP substitution of HA were detected using 1H-NMR, estimating a substitution efficiency of 10%-70%.

[0099] 1.2 Preparation of lipid nanogels encapsulating poly ICs

[0100] 1.2.1Microemulsion method

[0101] Microemulsions utilize nanoscale aqueous droplets in a water-in-oil system as the reaction system, thereby controlling the particle size of the nanoparticles formed therein. Figure 2 The specific steps are as follows:

[0102] 1) Using IGPAL-CO-520 as the surfactant and cyclohexane as the organic phase, an oil phase was prepared (IGEPAL-CO-520:cyclohexane = 3:7). A calcium phase was prepared by adding 150 μL of 500 mM CaCl2 and 50 μL of 2 mg / mL polyIC, and then slowly added dropwise to 7.5 mL of the oil phase. The system was sonicated for 1 min to ensure uniform dispersion.

[0103] 2) A phosphate phase was prepared by mixing 150 μL of 25 mM Na₂HPO₄ and 100 μL of 2 mg / mL HA-BP, and then slowly added dropwise to 7.5 mL of oil phase. The mixture was sonicated for 1 min to ensure uniform dispersion. The two water-in-oil systems were then mixed and incubated on a shaker at 180 rpm at room temperature for 30 min to form a nanogel composed of HA-BP and calcium phosphate mineralization.

[0104] 3) After the reaction is complete, add 15 mL of ethanol to disrupt the water-in-oil microemulsion, releasing the nanogel (NG). Collect the nanogel by centrifugation at 12000×g for 20 min. Disperse and resuspend the collected white precipitate in ethanol, and collect the nanogel by centrifugation at 12000×g for 10 min. Repeat the above steps once more to thoroughly wash away residual cyclohexane and IGEPAL-CO-520. Resuspend the nanogel in 400 μL of ultrapure water to obtain the nanogel. Sonicate the prepared nanogel and 400 μL of liposome shell for 15 s, and then squeeze it 6 times with a 400 nm cellulose membrane extruder to obtain a homogeneous and stable lipid nanogel encapsulating polyICs.

[0105] The results showed that the particle size of the nanogels prepared by this method, as measured by dynamic light scattering (DLS), was approximately 180 nm. Figure 3 B), the zeta potential is approximately -21mV. Figure 3 D).

[0106] 1.2.2 Microfluidic Method

[0107] 10 mg of phospholipids (3.85 mg cholesterol, 4.7 mg DLin-MC3-DMA, 0.95 mg DSPC, 0.5 mg DSPE-PEG2000) were dissolved in 300 μL of ethanol and mixed with 900 μL of citrate-sodium dihydrogen phosphate buffer solution containing 600 μg polyIC, 450 μg HA-BP, and pH 3-4 via a microfluidic chip. Dynamic light scattering (DLS, Malvern laser particle size analyzer (ZEN3690, UK)) revealed that the particle size of the prepared lipid nanogels was approximately 130 nm. Figure 3 A, Figure 3 B), the zeta potential is nearly neutral. Figure 3 D). Afterwards, dialyzing in flowing deionized water for 4-8 hours removed free ethanol and ions. Agarose gel electrophoresis results showed that the polyIC encapsulation rate in the lipid nanogel was 80%. Figure 3 E).

[0108] To verify whether phospholipid-encapsulated nanogels promote their stability in mouse bronchoalveolar lavage fluid (BALF), NG-pIC, LNP-pIC, and LNG-pIC were incubated in BALF at room temperature. The hydrated particle size of NG-pIC, LNP-pIC, and LNG-pIC after 2 h and 24 h of incubation was measured using a Malvern particle size analyzer (ZEN3690, UK). The results showed that although the particle size of LNP-pIC and LNG-pIC increased slightly, their particle size was uniform and their dispersibility was good. However, the particle size of NG-pIC increased from 180 nm to 580 nm after 2 h of incubation in BALF, and increased to 860 nm after 24 h. Figure 3 B, Figure 3 C).

[0109] To verify the protective effect of the lipid shell on polyICs, the formulation incubated in BALF for 24 hours was subjected to gel electrophoresis. The results showed that the polyICs in the bare gel core were degraded, while the polyICs encapsulated by phospholipids maintained their structural integrity. Figure 3 F), these results indicate that phospholipid-encapsulated nanogels of LNP-pIC and LNG-pIC can promote the stability of the gel core under physiological conditions and, in addition, protect poly ICs from degradation by nucleases in vivo.

[0110] Example 2: LNG-encapsulated polyICs are delivered to the lungs via nebulized inhalation APCs.

[0111] According to an embodiment of the present invention, in order to evaluate the in vivo delivery behavior of the LNG-pIC prepared in Example 1, LNG-pIC labeled with DiR (a deep red fluorescent probe for cell membranes) was delivered deep into the lungs of mice with B16F10 lung metastases via inhalation using a nebulizer. Various organs of the mice were harvested at different time points (1 h, 24 h, 48 h) for IVIS (in vivo imaging system for small animals). It was observed that the fluorescence emitted by DiR was mainly concentrated in the lungs. Further quantitative statistical analysis of the DiR fluorescence intensity in various organs showed that the DiR-labeled LNG-pIC mainly accumulated in the lung tissue, and the accumulation decreased over time. Figure 4 A-4C). Flow cytometry analysis was performed on lung tissue at different time points (1 h, 24 h, 48 h, and 96 h) to quantify the phagocytosis of LNG-pIC by alveolar macrophages (APCs) in the lungs. Results showed that 1 hour after inhalation of the DiR-labeled nanoparticles, DiR+ in alveolar macrophages (AMs) was approximately 28 ± 7%, in interstitial macrophages (IMs) approximately 17 ± 5%, and in dendritic cells (DCs) approximately 12 ± 3%. DiR+ levels in each cell type increased over 48 hours and then decreased. Figure 4 D). Simultaneously, flow cytometry analysis was performed on tumor-draining lymph nodes (TDLNs) at different time points (1h, 24h, 48h, and 96h). The results showed that DiR+mAMs, mCD103+DC, and mCD11b+DC in TDLNs increased within 48 hours and then decreased. Figure 4 E) indicates that LNG-pIC, which is engulfed by pulmonary APCs, migrates to TDLNs. These results suggest that inhaled LNG-pIC can be effectively taken up by pulmonary APCs and refluxed into TDLNs.

[0112] Example 3: Inhalation of LNG-pIC can effectively stimulate the production of type I interferon in mouse lung tissue.

[0113] According to an embodiment of the present invention, in order to detect whether LNG-pIC can induce higher levels of IFN-I in vivo, different formulations (pIC, NG-pIC, LNP-pIC, LNG-pIC) were delivered to the lungs via inhalation using a nebulizer for 24 hours. C57BL / B6 mice were euthanized, lung tissue homogenates were collected, the supernatant was obtained by centrifugation, and the IFN-β content was determined using an ELISA kit (InvivoGen, USA). Figure 5 A).

[0114] The results showed that, compared with the free polyIC group, NG group, and LNP-pIC group, inhaled LNG-pIC significantly increased the IFN-β secretion level in lung tissue, approximately three times that of LNP-pIC. Figure 5B) indicates that inhalation of LNG-pIC can induce higher levels of type I interferon in the lungs.

[0115] Example 4: Inhaled LNG-pIC can effectively treat lung metastases from B16F10 melanoma.

[0116] According to an embodiment of the present invention, in order to evaluate the therapeutic effects of different formulations on a mouse model of B16F10 melanoma lung metastasis, B16F10 melanoma cells (2 × 10⁻⁶) were used. 5 Lung metastasis model was established in C57BL / B6 mice by intravenous injection of B16F10 / mouse. Different preparations were administered on days 7 and 12 after modeling. On day 17, mice were euthanized, lung tissue was harvested, and lung metastases were counted and statistically analyzed. Results showed that the number of lung metastases in the LNG-pIC group was significantly reduced compared to the free polyIC group, NG group, and LNP-pIC group. Figure 6 A, Figure 6 B) indicates that inhalation of LNG-pIC can significantly improve the efficacy of anti-tumor treatment.

[0117] Example 5: Inhalation of LNG-pIC inhibits the growth of B16F10 melanoma lung metastases by increasing immune cells.

[0118] Experimental verification according to embodiments of the present invention revealed that, on day 18, mice were euthanized and lung tissue was extracted. Statistical analysis of lung metastases showed that LNG-pIC significantly inhibited the growth of B16F10 melanoma lung metastases. Flow cytometry analysis of lung tissue from the control and treatment groups, stained with CD80, CD86, CD4, CD8, and NK1.1, showed a significant increase in the proportion of CD80+, CD86+, CD8+, and IFN-γ+NK1.1+ cells in the LNG-pIC group. Figure 7 A- Figure 7 F), which is consistent with the in vivo treatment results, indicating that LNG-pIC can increase the number of immune cells, especially the proportion and number of CD8+ T cells, thereby exerting an anti-tumor effect.

[0119] Example 6: Inhaled LNG-pIC / siRNA combined therapy for lung metastases of B16F10 melanoma

[0120] According to an embodiment of the present invention, in order to evaluate the therapeutic effect of inhaled LNG-pIC / siRNA combined administration in a B16F10 melanoma lung metastasis model, B16F10 melanoma cells (2×10⁻⁶) were first subjected to treatment. 5Lung metastasis model was established by intravenous injection of B16F10 (10 mice) into C57BL / B6 mice. LNG-siRNA, LNG-pIC, and LNG-pIC / siRNA were administered by inhalation on days 7 and 12, respectively. On day 17, the mice were euthanized, lung tissue was removed, and lung metastases were counted and statistically analyzed.

[0121] The results showed that, compared with the untreated group, the LNG-siRNA group and the LNG-pIC group significantly inhibited the growth of lung metastases, while the number of lung metastases in the LNG-pIC / siRNA group was significantly reduced. Figure 8 A, Figure 8 B) indicates that the combined use of inhaled LNG-pIC / siRNA has a significant synergistic therapeutic effect.

[0122] To explore the related immune mechanism, the inventors intraperitoneally injected B16F10 melanoma lung metastasis model mice with anti-CD8 (2.43) / anti-CD4 (GK1.5) / anti-NK 1.1 (PK136). Mice injected with the antibodies inhaled the same dose of LNG-pIC / siRNA as the treatment group on days 7 and 12 after modeling. On day 20, the mice were euthanized and lung tissue was extracted. Figure 8 C), and perform statistical analysis on the count of lung metastases.

[0123] The results showed that, compared with the LNG-pIC / siRNA treatment group, the number of lung metastases in mice injected with anti-CD8 / anti-CD4 / anti-NK 1.1 was significantly increased, similar to that in the untreated control group. Figure 8 D), the results showed that a decrease in CD8 T cells, CD4 T cells, or NK cells would render their anti-tumor effects ineffective.

[0124] In summary, the combined treatment of LNG-pIC / siRNA has a very good effect on the B16F10 melanoma lung metastasis mouse model. However, the therapeutic effect completely disappeared after the reduction of CD8 T cells, CD4 T cells or NK cells, which further verifies that LNG-pIC can increase the number of immune cells and thus exert an anti-tumor effect.

[0125] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0126] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A liposome, characterized in that, include: Nanogel core and phospholipid layer; The phospholipid layer is wrapped around the outer surface of the nanogel core. The nanogel core includes a biodegradable anionic polymer and metal ions. The biodegradable anionic polymer is hyaluronic acid modified with bisphosphonate, and the metal ions are calcium ions. The hyaluronic acid modified with bisphosphonate is linked to the calcium ions through phosphate groups or carboxyl groups. The nanogel core further includes an adjuvant and siRNA, the adjuvant being used to activate T cells, and the adjuvant being present in the form of being inserted into the phospholipid layer or being completely encapsulated within the nanogel core.

2. The liposomes according to claim 1, characterized in that, The mass ratio of the biodegradable anionic polymer to metal ions is 0.45:

2.

3. The liposomes according to claim 1, characterized in that, The phospholipid layer includes at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids.

4. The liposomes according to claim 3, characterized in that, The phospholipid layer further includes cholesterol, PEG, or other auxiliary components.

5. The liposomes according to claim 1, characterized in that, The adjuvant includes any other component of a polyIC or TLR agonist.

6. The liposomes according to claim 5, characterized in that, Any other components of the TLR agonist include CpG, R837, MPLA, and their derivatives.

7. The liposomes according to claim 1, characterized in that, The nanogel core can carry 0.5%-10% adjuvant.

8. The liposomes according to claim 7, characterized in that, The adjuvant dosage is 4.6%.

9. The liposomes according to claim 1, characterized in that, The nanogel core can carry 0.5%-10% siRNA.

10. The liposomes according to claim 9, characterized in that, The amount of siRNA carried was 0.95%.

11. The liposome according to claim 9, characterized in that, The siRNA is used to interfere with the expression of immunosuppressive factors.

12. The liposomes according to claim 9, characterized in that, The siRNA includes at least one of PD-L1 siRNA, CTLA4 siRNA, and PD-1 siRNA.

13. The liposomes according to claim 12, characterized in that, The siRNA is selected from PD-L1 siRNA.

14. The method for preparing liposomes according to claim 1, characterized in that, include: A biodegradable anionic polymer is subjected to a crosslinking reaction in the presence of a crosslinking agent to obtain a nanogel core. The biodegradable anionic polymer is hyaluronic acid modified with bisphosphonic acid, and the crosslinking agent is calcium phosphate. The nanogel core is encapsulated in the presence of phospholipids to obtain the liposomes; The biodegradable anionic polymer and an adjuvant are subjected to the crosslinking reaction in the presence of a crosslinking agent to obtain the nanogel core.

15. The method according to claim 14, characterized in that, The mass ratio of the biodegradable anionic polymer, crosslinking agent, and adjuvant is 0.2:3:0.

1.

16. The method for preparing liposomes according to claim 1, characterized in that, include: The liposomes are obtained by mixing a biodegradable anionic polymer, phospholipids, a crosslinking agent, and an adjuvant. The biodegradable anionic polymer is hyaluronic acid modified with bisphosphonic acid, and the crosslinking agent is calcium phosphate.

17. The method according to claim 16, characterized in that, The mixing process is carried out in a microfluidic device.

18. The method according to claim 16, characterized in that, The mass ratio of the biodegradable anionic polymer, phospholipid, crosslinking agent, and adjuvant is 0.45:10:2:0.

675.

19. A pharmaceutical composition, characterized in that, include: The liposomes according to any one of claims 1 to 13.

20. The pharmaceutical composition according to claim 19, characterized in that, Further includes: Pharmaceutically acceptable carriers or excipients.

21. Use of the liposomes according to any one of claims 1 to 13 or the pharmaceutical composition according to any one of claims 19 to 20 in the preparation of a medicament for the treatment or prevention of melanoma.

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