Targeting Xkr8 in therapy

Abstract

The therapeutic system or combination includes a first therapeutic agent for treating a condition and a second therapeutic agent that can be administered within a predetermined time period of administration of the first therapeutic agent, where the second therapeutic agent inhibits the function of Xkr8.

Description

[Technical field]

[0001] [Government Interests] This invention was made with Government support under Grant No. CA223788 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 299,466, filed January 14, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0003] The following information is provided to aid the reader in understanding the technology disclosed below and the environment in which such technology may typically be used. Terms used herein are not intended to be limited to any particular narrow interpretation unless expressly stated otherwise herein. References listed herein may facilitate understanding of the technology or its background. The disclosures of all references cited herein are incorporated by reference.

[0004] Phosphatidylserine (PS) has been reported to be involved in the immunosuppression of cancer. As an important component of membrane structure, PS is restricted to the inner leaflet. This asymmetric distribution is maintained by the action of P4-ATPases such as ATP11A and ATP11C, which function as flippases in the plasma membrane to actively transfer PS from the outer leaflet to the inner leaflet. Scramblase breaks the membrane asymmetry, thereby randomizing all phospholipid species between the leaflets, effectively increasing the accumulation of PS on the outer side of the membrane.

[0005] The scramblase Xkr8 contains a caspase-3 recognition site in its C-terminal region and is generally believed to be post-transcriptionally regulated and activated by caspases during apoptosis.

[0006] Flippases are inactivated by caspases, resulting in irreversible exposure of PS on the cell surface, which is not limited to apoptosis but is also seen in coagulation, myoblast fusion, and T lymphocyte activation.

[0007] Macrophages phagocytose apoptotic cells but not live cells. PS serves as an important "eat me" signal. Binding of PS on apoptotic cells to its receptors on immune cells leads to severe immunosuppression. Moreover, this evolutionarily conserved mechanism is important for silent clearance of apoptotic cells in normal animal development. PS also functions as an upstream immune checkpoint that contributes to an immunosuppressed tumor microenvironment by preventing immune responses.

[0008] The discovery of PS receptors and their underlying signaling suggests a strategy for cancer immunotherapy by blocking immune suppressive pathways. However, such a strategy may also have a detrimental effect on T cell antitumor immunity. Blockade of PS via annexin A5 (AnxA5) or PS-specific antibodies (Abs) is another attractive strategy. A family of antibodies targeting PS has been developed. Unfortunately, despite the demonstrated efficacy and safety in preclinical and phase I / II clinical trials, larger phase III trials have failed to demonstrate efficacy over chemotherapy alone.

[0009] Another concern is the targeting of antibodies against other PS-expressing cells under physiological conditions and / or especially in combination with systemic chemotherapy due to comorbid diseases such as cardiovascular disease. Strategies that specifically target PS on tumor cells but do not affect PS on normal cells, and that prevent or reduce the amount of PS exposure when combined with other therapeutic / anticancer agents, may represent a more attractive approach. Summary of the Invention

[0010] In one embodiment, the therapeutic system or combination includes a first therapeutic agent for treating a condition and a second therapeutic agent administered within a predetermined time period of administration of the first therapeutic agent, the second therapeutic agent inhibiting the function of Xkr8. The first therapeutic agent may cause a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8. In some embodiments, the second therapeutic agent functions via RNA interference. The second therapeutic agent may, for example, include (e.g., in a complex) Xkr8 siRNA or may be Xkr8 siRNA. In some embodiments, the first therapeutic agent is a chemotherapeutic agent.

[0011] The therapeutic system may include a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers containing cationic groups, where a plurality of the first therapeutic agents, which are hydrophobic or lipophilic (e.g., hydrophilic or lipophilic chemotherapeutic agents), are integrated with the core of the nanostructure, and the second therapeutic agent, which includes Xkr8 siRNA, is added to the nanostructure and integrated with the cationic groups of the nanostructure. The nanostructure may further include a coating, application, or layer added to the nanostructure, which includes a negatively charged targeting agent (i.e., an agent that actively targets an area of ​​interest, such as a tumor). The coating, application, or layer need not be continuous. The negative charge provides an anchoring interaction via charge-charge interaction with the cationic groups of the amphiphilic polymers forming the nanostructure. In addition to providing targeting, such negatively charged agents aid in charge neutralization / shielding of positive charges to achieve a nanostructure / nanocarrier that exhibits near charge neutrality. The coating, application or layer may further comprise a hydrophilic polymeric compound, which may, for example, comprise a negative charge to anchor the hydrophilic polymeric compound to the cationic groups via charge-charge interactions, which may provide further charge neutralization and, in some embodiments, may provide a degree of shielding for the targeting agent.

[0012] In some embodiments, the negatively charged targeting agent includes or is a ligand, peptide, aptamer, polysaccharide, or antibody to a cell receptor. The negative charge may be intrinsic to the targeting agent or may be added to the targeting agent (e.g., via conjugation with a negatively charged molecule or compound). In some representative embodiments, the targeting agent is a negatively charged CD44 ligand. In some embodiments, the application applied to the nanostructure includes a negatively charged receptor ligand, such as a negatively charged CD44 ligand, and a hydrophilic polymer. The hydrophilic polymer may include a negative charge as described above. In some embodiments, the hydrophilic polymer includes a conjugate of a negatively charged molecule or compound with a hydrophilic polymer, or is a conjugate of a negatively charged molecule or compound with a hydrophilic polymer. The negatively charged molecule or compound conjugated to the hydrophilic polymer may be a targeting agent (e.g., a CD44 ligand).

[0013] The CD44 ligand may include, for example, osteopontin, collagen, matrix metalloproteases, chondroitin sulfate, hyaluronic acid, or derivatives thereof. In some embodiments, the CD44 ligand is chondroitin sulfate.

[0014] In some embodiments, the second therapeutic agent, including Xkr8 siRNA, is added to the nanostructure before the negatively charged targeting agent (e.g., a negatively charged CD44 ligand) and (in embodiments in which the application agent further comprises the hydrophilic polymeric compound) the hydrophilic polymeric compound are applied. The targeting agent and the hydrophilic polymeric compound may be added as a mixture.

[0015] The first therapeutic agent may be a small molecule therapeutic compound, for example having a molecular weight of less than 1 kDa.

[0016] As used herein, the term "cationic group" refers to a group that is cationic in nature or that forms a cation in vivo. In some embodiments, the group that forms a cation in vivo is an amine group, and the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. In some embodiments, the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

[0017] The hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides, hi some embodiments, the hydrophilic polymer is polyethylene glycol.

[0018] The ratio of the negatively charged targeting agent to the hydrophilic polymer added to the nanostructure may be determined such that uptake of the nanostructure in one or more regions other than the region of interest is maintained at a sufficiently low level to allow interaction with the negatively charged targeting agent in the region of interest (e.g., a tumor). In some embodiments, the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is determined such that uptake of the nanostructure in the liver of a patient is maintained at a sufficiently low level to allow interaction of CD44 with the negatively charged CD44 ligand in a tumor distant from the liver.

[0019] In some embodiments, each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer. The hydrophobic polymer backbone may further comprise a pendant lipid group. The hydrophobic polymer backbone may be formed via free radical polymerization. The hydrophobic polymer backbone may be formed via reversible-deactivation radical polymerization.

[0020] In another aspect, a method of providing a combination therapy for treating a condition includes administering a first therapeutic agent for treating a condition and administering a second therapeutic agent within a predetermined time period after administering the first therapeutic agent, where the second therapeutic agent inhibits expression or function of Xkr8. The first therapeutic agent may cause a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8. In some embodiments, the second therapeutic agent functions via RNA interference. The second therapeutic agent may include or be an Xkr8 siRNA. In some embodiments, the first therapeutic agent is a chemotherapeutic agent.

[0021] The combination therapy may be delivered via a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups, wherein the plurality of first therapeutic agents, which are hydrophobic or lipophilic, are integrated with the core of the nanostructure, and the second therapeutic agent, which comprises Xkr8 siRNA, is added to the nanostructure and integrated with the cationic groups of the nanostructure. The nanostructure may further comprise a coating, application or layer applied to the nanostructure, the application comprising a negatively charged targeting compound (e.g., CD44 ligand) as described above. The application may further comprise a hydrophilic polymeric compound. The hydrophilic polymeric compound may comprise a negative charge. In some embodiments, the hydrophilic polymeric compound comprises a complex of a negatively charged molecule or compound with a hydrophilic polymer. In some embodiments, the negatively charged targeting agent is a CD44 ligand, and the negatively charged molecule or compound complexed with the hydrophilic polymer may be a CD44 ligand. The CD44 ligand can be, for example, osteopontin, collagen, matrix metalloprotease, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

[0022] In a further aspect, the formulation comprises a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers, a plurality of first hydrophobic or lipophilic therapeutic agents integrated with the core of each of the nanostructures, and a second therapeutic agent that inhibits the expression or function of Xkr8. The plurality of amphiphilic polymers may, for example, comprise cationic groups. The second therapeutic agent may, for example, comprise or be Xkr8 siRNA that is added to the nanostructure so as to be integrated with the cationic groups of the nanostructure. The nanostructure may further comprise a coating, application or layer that is added to the nanostructure, the application comprising a negatively charged targeting agent as described above. The application may further comprise a hydrophilic polymeric compound. As described above, the hydrophilic polymeric compound may comprise a negative charge. In some embodiments, the hydrophilic polymeric compound comprises a complex of a negatively charged molecule or compound with a hydrophilic polymer. The negatively charged molecule or compound complexed with the hydrophilic polymer may, for example, be a targeting agent (e.g., CD44 ligand). As further described above, the CD44 ligand herein may include, for example, osteopontin, collagen, matrix metalloproteinase, chondroitin sulfate, hyaluronic acid, or derivatives thereof. In some embodiments, the CD44 ligand is chondroitin sulfate. The second therapeutic agent, including Xkr8 siRNA, may be added to the nanostructure before the negatively charged targeting agent, such as CD44 ligand, is applied, and before the negatively charged targeting agent and hydrophilic polymer are combined (in embodiments in which the application further includes a hydrophilic polymer).

[0023] The first therapeutic agent may be, for example, a small molecule therapeutic compound, hi some embodiments, the first therapeutic agent has a molecular weight of less than 1 kDa.

[0024] As mentioned above, the cationic group may be an intrinsically cationic group or a group that forms a cation in vivo. In some embodiments, the group that forms a cation in vivo is an amine group, and the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. In some embodiments, the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

[0025] In some embodiments, the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides, hi some embodiments, the hydrophilic polymer is polyethylene glycol.

[0026] The ratio of the negatively charged targeting agent to the hydrophilic polymer added to the nanostructure can be determined such that uptake of the nanostructure in one or more regions other than the region of interest is maintained at a sufficiently low level to allow interaction of the negatively charged targeting agent in the region of interest (e.g., a tumor). In some embodiments, the ratio of the negatively charged CD44 ligand to the hydrophilic polymer can be determined such that uptake of the nanostructure in the liver of a patient is maintained at a sufficiently low level to allow interaction of the negatively charged CD44 ligand with CD44 in a tumor distant from the liver.

[0027] In some embodiments, each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer. The hydrophobic polymer backbone may further comprise a pendant lipid group. In some embodiments, the hydrophobic polymer backbone is formed via free radical polymerization. In some embodiments, the hydrophobic polymer backbone is formed via controlled / living radical polymerization or reversible deactivation radical polymerization.

[0028] In yet another aspect, a method of providing therapy to a patient includes administering a first therapeutic agent for treating a condition and administering a second therapeutic agent delivered within a predetermined time period after administering the first therapeutic agent, the second therapeutic agent inhibiting expression or function of Xkr8. The first therapeutic agent may, for example, cause a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8. In some embodiments, the second therapeutic agent functions via RNA interference. The second therapeutic agent may, for example, include or be an Xkr8 siRNA. In some embodiments, the first therapeutic agent is a chemotherapeutic agent.

[0029] In some embodiments, the first and second therapeutic agents are administered via a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers containing cationic groups, the first therapeutic agents, which are hydrophobic or lipophilic, are associated with the core of the nanostructure, and the Xkr8 siRNA is added to the nanostructure and associated with the cationic groups of the nanostructure. The first and second therapeutic agents may be delivered in a formulation, for example, as described above and elsewhere herein.

[0030] The devices, systems, methods and compositions of the present invention, together with their attributes and attendant advantages, will best be appreciated and understood in consideration of the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0031] [Figure 1a] FIG. 1a illustrates the volcano plot for RNA-seq analysis of CT26 tumors in mice treated with FuOXPNPs versus empty NPs.

[0032] [Figure 1b] Figure 1b illustrates qRT-PCR analysis of mXkr8 mRNA expression in CT26 tumors of mice treated with empty NPs or FuOXPNPs. n=5. ns indicates non-significant p>0.05. *** indicates p<0.001.

[0033] [Figure 1c] Figure 1c illustrates qRT-PCR analysis of Xkr8 mRNA expression in various types of cultured tumor cells 24 hours after treatment with FuOXP, DOX, and PTX, respectively. n=3. *** indicates p<0.001.

[0034] [Figure 1d] Figure 1d illustrates Western analysis of hXkr8 protein expression in PANC-1 or HT29 tumor cells 24 hours after treatment with FuOXP, DOX, and PTX, respectively. The Mw of hXkr8 is 45 kDa. The Mw of β-tubulin is 55 kDa.

[0035] [Figure 1e] Figure 1e illustrates densitometric analysis of the protein bands in Figure 1d. n=3. *** indicates p<0.001.

[0036] [Figure 1f]FIG. 1f illustrates qRT-PCR analysis of Xkr8 mRNA expression over time in CT26 tumor cells treated with FuOXP for 12 hours followed by actinomycin D for 2 or 4 hours. n=3. ns indicates not significant. [Figure 1g] Figure 1g illustrates qRT-PCR analysis of Xkr8 mRNA expression over time in PANC-1 tumor cells treated with FuOXP for 12 hours followed by actinomycin D for 2 or 4 hours. n=3. ns indicates not significant.

[0037] [Figure 1h] Figure 1h illustrates the change in Xkr8 mRNA expression in CT26. n=3. *** indicates p<0.001. [Figure 1i] Figure 1i illustrates the change in Xkr8 mRNA expression in PANC-1. n=3. *** indicates p<0.001. [Figure 1j] FIG. 1j illustrates the change in (human) Xkr8 protein expression in PANC-1 cells treated with N-acetylcysteine ​​(NAC) for 12 hours followed by treatment with different drugs for an additional 12 hours. [Figure 1k] Figure 1k illustrates the change in (human) Xkr8 protein expression in PANC-1 cells treated with N-acetylcysteine ​​(NAC) for 12 hours and then with different drugs for another 12 hours, where Figure 1k illustrates the densitometric analysis of the protein bands in the Western blot (Figure 1j). n=3. *** indicates p<0.001.

[0038] [Figure 2a] FIG. 2a illustrates a schematic diagram of an embodiment of a protocol for preparing FuOXP / siRNA co-loaded PMBOP-CPNPs.

[0039] [Figure 2b] FIG. 2b illustrates the CMC of the PMBOP polymer.

[0040] [Figure 2c] Figure 2c illustrates the biophysical characterization of PMBOP / FuOXP mixed micelles at different carrier / drug ratios (w / w). n=3. Results are shown as mean ± SEM.

[0041] [Figure 2d] FIG. 2d illustrates the gel retardation assay of PMBOP / FuOXP / siRNA complexes at various N / P ratios.

[0042] [Figure 2e] FIG. 2e illustrates the size and zeta potential of PMBOP / FuOXP / siRNA complexes at various N / P ratios.

[0043] [Figure 2f] FIG. 2f illustrates the size and zeta potential of FuOXP / siRNA co-loaded PMBOP-CPNPs (coated with CS alone) at various N / P / S ratios.

[0044] [Figure 2g] Figure 2g illustrates the size and zeta potential of FuOXP / siRNA co-loaded PMBOP-CPNPs (coated with a mixture of CS and PEG-CS) at various N / P / S(CS) / S(PEG-CS) ratios, n=3 (Figures 1e-f).

[0045] [Figure 2h] Figure 2h illustrates the spherical shape of siRNA-coloaded and non-coloaded PMBOP-CP NPs by cryo-electron microscopy. Scale bar indicates 100 nm.

[0046] [Figure 2i] Figure 2i illustrates the cumulative FuOXP release graph from PMBOP-CPNPs with or without siRNA complexes in PBS or mouse serum. n=3. * indicates p<0.05. ** indicates p<0.01.

[0047] [Figure 2j] FIG. 2j illustrates the protection of siRNA in PMBOP-CPNP against digestion by RNase.

[0048] [Figure 3a] FIG. 3a illustrates the fluorescence microscopy examination of tumor and liver sections 24 h after iv injection of PMBOP NPs loaded with Cy5.5-siXkr8 at various N / P / S(CS) / S(PEG-CS) ratios. [Figure 3b] Figure 3b illustrates the fluorescence microscopy of tumor and liver sections 24 h after iv injection of PMBOP NPs loaded with Cy5.5-siXkr8 at various N / P / S(CS) / S(PEG-CS) ratios.

[0049] [Figure 3c-3d] FIG. 3c illustrates NIR whole body imaging of CT26 tumor-bearing mice 24 hours after iv administration of Cy5.5-siXkr8-loaded PMBOP-CPNP.

[0050] FIG. 3d illustrates ex vivo imaging of (CT26) tumors and major organs 24 hours after iv administration of Cy5-siXkr8-loaded PMBOP-CPNP.

[0051] [Figure 3e] Figure 3e shows the fluorescence intensity of (Cy5.5-siXkr8) in tumor and liver at various times after iv administration of Cy5.5-siXkr8-loaded PMBOP-CPNP. n=3. ** indicates p<0.01. *** indicates p<0.001.

[0052] [Figure 3f]Figure 3f illustrates the change in (Cy5.5-siXkr8) fluorescence intensity in blood at various times after iv administration of free Cy5.5-siXkr8 or PMBOP-CPNP loaded with Cy5.5-siXkr8. n=3. *** indicates p<0.001. [Figure 3g] Figure 3g illustrates the change in (Cy5.5-siXkr8) fluorescence intensity in blood at various times after iv administration of free Cy5.5-siXkr8 or PMBOP-CP NPs loaded with Cy5.5-siXkr8. n=3. *** indicates p<0.001.

[0053] [Figure 3h] FIG. 3h illustrates ex vivo imaging of tumors and major organs 24 hours after iv administration of Cy5.5-siXkr8-loaded PMBOP-CPNPs in various types of sc tumor models. [Figure 3i] FIG. 3i illustrates ex vivo imaging of tumors and major organs 24 hours after iv administration of Cy5.5-siXkr8-loaded PMBOP-CPNPs in a CT26 orthotopic model.

[0054] [Figure 3j] FIG. 3j shows the CT26 lung metastasis model.

[0055] [Figure 3k] Figures 3k-n show confocal laser scanning microscopy images of tumor (scCT26) sections 24 hours after iv administration of Cy5.5siXkr8-loaded PMBOP-CP NPs. Figure 3k shows a composite image of cell nuclei (Hoechst), F-actin (AlexaFluor®488 Phalloidin) and siRNA (Cy5.5) magnified at 20x. Scale bar indicates 30 μm. [Figure 3l] Figure 3l illustrates 3D Z-stacking of two layers of 2D scan images from different depths. Magnification 600x. Scale bar indicates 1 μm. Magnification 600x. [Figure 3m] Figure 3m: Green: 0.1 μm layer of the first scan. Scale bar indicates 1 μm. Magnification 600x. Large white arrow indicates spotty pattern. Small white arrow indicates diffuse pattern. [Figure 3n] FIG. 3n, Red: 0.5 μm layer from the first scan. Scale bar indicates 1 μm. Magnification 600x. Large white arrow indicates spotty pattern. Small white arrow indicates diffuse pattern.

[0056] [Figure 4a-4c] Figures 4a and 4b illustrate NIR whole-body imaging (Figure 4a) and ex vivo imaging (Figure 4b) of CT26 tumor targeting in WT and CD44- / - mice 24 hours after iv administration of PMBOP-CPNP loaded with Cy5.5-siXkr8.

[0057] FIG. 4c shows CT26 tumor-bearing WT mice and CD44 - / - Illustrates ex vivo imaging of blood taken from mice.

[0058] [Figure 4d] Figure 4d illustrates (Cy5.5-siXkr8) fluorescence intensity in tumors and liver 24 hours after iv administration of NPs. n=3. *** indicates p<0.001. [Figure 4e] Figure 4e illustrates (Cy5.5-siXkr8) fluorescence intensity in blood 24 hours after iv administration of NPs. n=3. *** indicates p<0.001.

[0059] [Figure 4f] FIG. 4f illustrates NIR whole-body imaging of CT26 tumor-bearing zombie mice 24 hours after iv administration of NPs.

[0060] [Figure 4g]Figure 4g illustrates (Cy5.5-siXkr8) fluorescence intensity in tumors and liver 24 hours after iv administration of NPs. n=3. *** indicates p<0.001.

[0061] [Figure 4h] Figure 4h illustrates flow analysis of CD44 expression in (WT and CD44- / -) mouse LSEC, Tq, Ta, HUVECq, HUVECa and CT26 cells. n=3. * indicates p<0.05. *** indicates p<0.001.

[0062] [Figure 4i] Figure 4i illustrates the quantitative analysis of % and MFI of Cy5.5+ cells after 4 hours of incubation of Cy5.5-siXkr8-loaded PMBOP-CPNPs with (WT and CD44- / -) LSEC, HUVECq and HUVECa cells, respectively. n=3. ** indicates p<0.01.

[0063] [Figure 4j] Figure 4j illustrates flow analysis of Cy5.5+CT26 cells (lower chamber) 12 hours after addition of PMBOP-CPNP loaded with Cy5.5-siXkr8 to HUVECs (upper chamber) in a Transwell plate. n=3. *** indicates p<0.001.

[0064] [Figure 4k] Figure 4k illustrates the quantitative analysis of cellular uptake of PMBOP-CPNP loaded with Cy5.5-siXkr8 by CT26, HUVECa, Tq and Ta cells. n=3. *** indicates p<0.001.

[0065] [Figure 4l]Figure 4l illustrates an embodiment of the proposed model of PMBOP-CP NPs mediated tumor targeting through both EPR and transcytosis. NPs coated with CS alone were rapidly cleared by LSEC / CD44-mediated liver uptake, with limited accumulation in tumors. Addition of an optimized amount of CS / PEG-CS to the NPs significantly reduced LSEC uptake without significantly affecting the interaction with tumor ECs, resulting in effective tumor targeting.

[0066] [Figure 5a] Figure 5a illustrates the ICP-MS analysis of plasma concentrations of Pt after tail vein injection of free FuOXP / siXkr8 or FuOXP / siXkr8NP in naive BALB / c mice. The doses were 5 mg / kg for FuOXP and 1 mg / kg for siXkr8, respectively. n=3. *** indicates p<0.001.

[0067] [Figure 5b] Figure 5b illustrates the pharmacokinetic parameters of Pt analyzed by a non-compartmental model. n=3. Results are shown as mean ± SEM.

[0068] [Figure 5c] Figure 5c illustrates the biodistribution of Pt in various organs at various times after iv injection of FuOXP / siXkr8NP in BALB / c mice bearing CT26 tumors. n=3. ** indicates p<0.01. *** indicates p<0.001. [Figure 5d] Figure 5d illustrates the biodistribution of Pt in various organs at different times after iv injection of FuOXP / siXkr8 NPs in BALB / c mice bearing CT26 tumors. n=3. ** indicates p<0.01. *** indicates p<0.001.

[0069] [Figure 5e]Figure 5e illustrates qRT-PCR analysis of plasma concentrations of siXkr8 after iv injection of free FuOXP / siXkr8 or FuOXP / siXkr8NP in naive BALB / c mice. n=3. *** indicates p<0.001.

[0070] [Figure 5f] Figure 5f illustrates that the pharmacokinetic parameters of siXkr8 were analyzed by a non-compartmental model. n = 3. Results are presented as mean ± SEM.

[0071] [Figure 5g] Figure 5g illustrates the biodistribution of siXkr8 in various organs at various times after iv injection of FuOXP / siXkr8NP in BALB / c mice bearing CT26 tumors. n=3. * indicates p<0.05. *** indicates p<0.001. [Figure 5h] Figure 5h illustrates the biodistribution of siXkr8 in various organs at different times after iv injection of FuOXP / siXkr8 NPs in BALB / c mice bearing CT26 tumors. n=3. * indicates p<0.05. *** indicates p<0.001.

[0072] [Figure 5i] FIG. 5i illustrates the fluorescence microscopy images of cultured CT26 tumor cells 2 hours after treatment with Cy5.5-siXkr8-loaded PMBOP-CPNPs.

[0073] [Figure 5j] Figure 5j illustrates the efficiency of siRNAPMBOP-CP NP-mediated gene knockdown in cultured MC38-Luc cells. MC38-Luc cells were pretreated with various endocytosis inhibitors for 1 h and then treated with luciferase siRNA NPs. After 24 h, luciferase activity was examined in the variously treated cells. n=3. Results are shown as mean ± SEM.

[0074] [Figure 5k] Figure 5k illustrates qRT-PCR analysis of Xkr8 mRNA expression levels in CT26 cells 24 hours after treatment with siCT NP, siXkr8Lipo (Lipofectamine RNAiMAX Gene Delivery Reagent), or siXkr8 NP. n=3. * indicates p<0.05. *** indicates p<0.001.

[0075] [Figure 5l] FIG. 5l illustrates MC38-Luc tumor-bearing C57BL / 6 mice administered a 2 mg / kg dose of luciferase siRNA NPs iv once every 5 days and were subjected to whole-body bioluminescence imaging the day after each treatment. n=3. ns indicates not significant. *** indicates p<0.001.

[0076] [Figure 5m] Figure 5m illustrates CT26 tumor-bearing mice treated with FuOXP at a dose of 5 mg / kg and siRNA at a dose of 1 mg / kg, with siXkr8NPs or with FuOXP / siXkr8 co-loaded NPs once every 5 days. Xkr8 mRNA expression in tumors was examined by qRT-PCR the day after the final treatment. n=5. * indicates p<0.05. *** indicates p<0.001.

[0077] [Figure 6a] Figure 6a illustrates a study of CT26 tumor cells treated with control or siXkr8NPs for 72 hours followed by FuOXP / siRNA co-loaded NPs. The number of Annexin V+ cells was examined by flow cytometry after 24 hours. n=3. *** indicates p<0.001.

[0078] [Figure 6b]Figure 6b illustrates a study of CT26 tumor cells that underwent similar treatment as described in Figure 6a and the amount of secreted Annexin V+ EVs was examined by flow cytometry. n=3. ** indicates p<0.01. *** indicates p<0.001.

[0079] [Figure 6c] Figure 6c illustrates a study of CT26 tumor cells in which tumor cells were cultured with primary macrophages after undergoing treatment similar to that described in Figure 6a. The percentage of M1 and M2 cells was then quantified based on flow analysis of various M1 / M2 surface markers. n=3. ** indicates p<0.01.

[0080] [Figure 6d] Figure 6d illustrates a study of mice bearing CT26 tumors receiving various treatments with siRNA at a dose of 1 mg / kg and FuOXP at a dose of 5 mg / kg, once every 5 days, for three times. Tumor burden was followed once every 2 days. n=5. ** indicates p<0.01. *** indicates p<0.001. [Figure 6e] Figure 6e illustrates a study of mice bearing Panc02 tumors that received various treatments with siRNA at a dose of 1 mg / kg and FuOXP at a dose of 5 mg / kg, once every 5 days, for three times. Tumor burden was followed once every 2 days. n=5. ** indicates p<0.01. *** indicates p<0.001.

[0081] [Figure 6f] Figures 6f-6k illustrate a study of mice bearing CT26 tumors that received different treatments as described in Figure 6d. At the end of the treatment study, single cell suspensions were prepared and subjected to different flow analyses including Annexin V+ cells. n=5. * indicates p<0.05. *** indicates p<0.001. [Figure 6g] At the end of the treatment study, single cell suspensions were prepared and subjected to various flow analyses including CD45+ cells. n=5. *** indicates p<0.001. [Figure 6h]At the end of the treatment study, single cell suspensions were prepared and subjected to various flow analyses including regulatory T cells. n=5. ** indicates p<0.01. [Figure 6i] At the end of the treatment study, single cell suspensions were prepared and subjected to various flow analyses including M1 / M2-like ratio. n=5. ** indicates p<0.01. [Figure 6j] At the end of the treatment study, single cell suspensions were prepared and subjected to various flow analyses including IFNγ+CD8+ cells, n=5. ns indicates non-significant p>0.05. [Figure 6k] At the end of the treatment study, single cell suspensions were prepared and subjected to various flow analyses including GzmB CD8+ cells, n=5. ns indicates non-significant p>0.05.

[0082] [Figure 6l] Figure 6l illustrates a study of mice bearing Panc02 tumors that received various treatments as described in Figure 6e. At the end of the treatment study, single cell suspensions were prepared and subjected to flow analysis of PD-1+CD8+ cells. n=5. ** indicates p<0.01.

[0083] [Figure 6m] Figure 6m and Figure 6n illustrate a study of mice bearing Panc02 tumors that received various treatments at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg for siXkr8, FuOXP, and anti-PD-1, respectively, once every 5 days for three times when tumors reached a size of approximately 155 mm3. In Figure 6m, tumor growth was followed. n=8. ** indicates p<0.01. *** indicates p<0.001. [Figure 6n] In Figure 6n, survival was followed. n=8. ** indicates p<0.01. *** indicates p<0.001.

[0084] [Figure 7a] Figure 7a illustrates a study of CT26 tumor-bearing mice that received the treatment described in Figure 6d. Mice were weighed once every two days.

[0085] [Figure 7b] Figure 7b shows serum levels of AST and ALT at the end of the treatment study. n=3. ns indicates non-significant p>0.05.

[0086] [Figure 7c] FIG. 7c illustrates the histology of major organs in mice receiving the various treatments described in FIG. 6d.

[0087] [Figure 7d] Figure 7d illustrates serum levels of TNF-α and IL-6 2 hours after iv administration of siRNAPMBOP-CP NPs or siRNA complexed with DOTAP liposomes (N / P, 10 / 1) at a dose of 1 mg / kg siRNA. n=3. ns indicates non-significant p>0.05. *** indicates p<0.001.

[0088] [Figure 7e] FIG. 7e illustrates that FuOXPNP caused minimal changes in Xkr8 mRNA levels in the liver. [Figure 7f] FIG. 7f illustrates that FuOXP NPs caused minimal changes in PS+ cells in the liver.

[0089] [Figure 7g] Figure 7g illustrates that minimal changes in CD45+ cells in the liver were caused by FuOXP / siXkr8NP. n=3. ns indicates not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] It will be readily understood that the components of the embodiments as generally described and illustrated in the Figures herein may be arranged and designed in a wide variety of different configurations in addition to the exemplary embodiments described. Thus, the following more detailed description of the exemplary embodiments as illustrated in the Figures is not intended to limit the scope of the embodiments as claimed, but merely to illustrate exemplary embodiments.

[0091] References throughout this specification to "one embodiment" or "an embodiment" (or the like) mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, although phrases such as "in one embodiment" or "in an embodiment" appear in various places throughout this specification, they are not necessarily all referring to the same embodiment.

[0092] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided in order to provide a thorough understanding of the embodiments. However, one of ordinary skill in the relevant art will recognize that various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0093] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a therapeutic agent" includes a plurality of such therapeutic agents known to those of skill in the art, and a reference to "the therapeutic agent" is a reference to one or more such therapeutic agents known to those of skill in the art, and equivalents thereof. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each discrete value falling within the range. Unless otherwise indicated herein, and each discrete value, as well as intermediate ranges, are incorporated herein as if individually set forth herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or expressly contraindicated by the text.

[0094] In some embodiments herein, a first therapeutic agent for treating a pathology is administered or delivered, and a second therapeutic agent is administered or delivered within a predetermined time (including at the same time, contemporaneous time, or different time) of the first therapeutic agent. The second therapeutic agent inhibits the expression or function of Xkr8. The second therapeutic agent may inhibit the expression or function of Xkr8, for example, at the genome level, the mRNA level, or the protein level. Gene silencing therapy may be used either during transcription or during translation, for example, via small molecule therapy, nucleotide-based therapy, CRISPR, etc. In post-transcriptional silencing, RNA interference (RNAi) may be provided or achieved, for example, using small molecules, microRNA (miRNA) and / or small interfering RNA (siRNA). Antisense oligonucleotides may be used to target mRNA. Similarly, ribozymes may be used to target mRNA. Protein level therapy may be provided or achieved, for example, using small molecules and degradation of the target protein. The first therapeutic agent may, for example, cause a therapeutic response that induces the expression of Xkr8 in addition to activating Xkr8. For example, studies herein have found that treating tumors with chemotherapeutic drugs significantly upregulates Xkr8 mRNA in vitro and in vivo, suggesting that Xkr8 may be a new therapeutic target for cancer treatment. In the case of combined treatment in which a first therapeutic agent is delivered to treat a pathology and a second therapeutic agent is delivered to inhibit Xkr8 expression or function, the second therapeutic agent may be delivered within a time period determined to provide inhibition of upregulation / activation due to the therapeutic effect of the first therapeutic agent. In some embodiments, the first therapeutic agent and the second therapeutic agent are delivered via a common carrier, which may be a nanostructure-based carrier or a nanocarrier.Various nanocarriers based on self-assembling amphiphilic polymers are described, for example, in U.S. Patent Nos. 10,172,795 and 9,855,341, U.S. Patent Publication Nos. 2018 / 0214563 and 2021 / 0236645, and PCT International Patent Application No. PCT / US22 / 43938, filed September 19, 2022, the disclosures of which are incorporated herein by reference. Depending on the nature of the first and second therapeutic agents, one skilled in the art can select or design an appropriate carrier.

[0095] In some embodiments herein, a novel nanocarrier capable of co-delivering siRNA (and / or other polynucleotide (e.g., nucleic acid such as DNA, RNA, etc.)-based therapeutics) and small molecule drugs was developed. The nanocarrier was extensively characterized in terms of tumor targeting efficiency and the underlying mechanism. Furthermore, the therapeutic effect and the underlying mechanism of co-delivery of siXkr8 and representative anticancer drugs 5-Fu / oxaliplatin were investigated in syngeneic mouse models of colon and pancreatic cancer.

[0096] In an independent study to clarify the mechanism of chemotherapy resistance in colorectal cancer (CRC), RNA-seq was performed to examine the changes in gene expression profiles after treatment of CT26 tumor-bearing mice with FuOXP. FuOXP is a prodrug conjugate of 5-FU and oxoplatin that has been previously reported to improve antitumor activity and reduce cytotoxicity against normal cells. CT26 is an isogenic CRC model that responded poorly to moderately to 5-FU / OXP, similar to FuOXP. As shown in Figure 1a, Xkr8 was one of the genes with the most significant increase in mRNA expression. The induction of mouse Xkr8 (murine Xkr8: mXkr8) mRNA expression in vivo was further confirmed by qRT-PCR (Figure 1b). Similar to FuOXP, induction of Xkr8 mRNA expression by two other drugs (doxorubicin (DOX) and paclitaxel (PTX)) was also demonstrated in cultured mouse CRC (CT26) and human CRC (HT-29) cell lines, and pancreatic cancer (PCa) (Panc02, PANC-1) cell lines, respectively (Fig. 1c). Induction of human Xkr8 (hXkr8) was confirmed at the protein level after treatment with the three drugs in human CRC (HT-29) and PCa (PANC-1) cell lines, respectively (Fig. 1d and Fig. 1e). Induction of mXkr8 at the protein level could not be evaluated in mouse cancer cells in vitro and in vivo as a result of the lack of available antibodies.

[0097] Figure 1f shows the kinetics of mXkr8 mRNA levels in CT26 cells after 12 h of FuOXP treatment followed by an additional 2 h of treatment with the transcription inhibitor actinomycin D (ActD). In cells treated with ActD alone, the level of mXkr8 mRNA gradually decreased, reflecting the normal degradation rate of Xkr8 mRNA in CT26 cells. Cells treated with FuOXP for 12 h showed high initial levels of mXkr8 mRNA, consistent with the data shown in Figure 1c. Nevertheless, cells receiving the combination treatment showed similar kinetics of decrease in the level of mXkr8 mRNA, suggesting that FuOXP does not affect the degradation rate of mXkr8 mRNA. Similar results were observed in PANC-1 cells (Figure 1g). This data suggested that FuOXP, and potentially other drugs as well, caused Xkr8 induction through increased gene transcription.

[0098] The mechanism of Xkr8 induction by various chemotherapy drugs has not been fully elucidated. Preliminary studies suggested that pretreatment with the antioxidant N-acetylcysteine ​​(NAC) partially attenuated Xkr8 induction by the three drugs at both the mRNA level (Fig. 1h-i) and protein level (Fig. 1j-k) in CT26 cells (Fig. 1h) and PANC-1 cells (Fig. 1i-k). Without being limited to any mechanism, these results suggest a role for oxidative stress in Xkr8 induction. This observation is consistent with previous reports that oxidative stress may be involved in the exposure of PS in tumor vasculature.

[0099] In some studies herein, Xkr8 expression is reduced by siRNA-mediated knockdown.Furthermore, representative nanocarrier PMBOP-CP has been developed to achieve co-delivery of siXkr8 and FuOXP.Nanocarriers suitable for use herein, including PMBOP-CP, have been described in PCT International Patent Application No. PCT / US22 / 43938, which is assigned to the assignee of the present application.

[0100] Figure 2a illustrates the main components and steps in the development of PMBOP-CP nanocarriers. PMBOP is an amphiphilic cationic polymer that self-assembles to form micelles in aqueous solution. The 1-octadecene lipid motif in the PMBOP polymer is expected to help improve the loading of FuOXP into the hydrophobic / lipophilic core while promoting the interaction with the cell membrane to improve gene transfer. The biguanidine motif was designed to enhance the interaction with siRNA as a result of its highly cationic nature. The synthetic route of PMBOP is shown in Scheme 1 (Figure 3a) of PCT International Patent Application No. PCT / US22 / 43938, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. 1 This was confirmed by HNMR spectroscopy.

[0101] The PMBOP polymer had a low CMC of 0.0033 mg / mL (Fig. 2b) and readily formed micelles with a size of 173.2 nm in PBS. FuOXP could be loaded into PMBOP micelles at a carrier / drug weight ratio as low as 2 / 1 (Fig. 2c). Both drug-free and FuOXP-loaded PMBOP micelles readily formed complexes with siRNA. Gel retardation assays showed that siRNA was effectively incorporated into the micelles at nitrogen / phosphate (N / P) ratios of 1 or higher (Fig. 2d). At an N / P ratio of 10 / 1, the resulting PMBOP / FuOXP / siRNA complexes were positively charged (+17.5 mV) and had a size of 107.5 nm, smaller than that of PMBOP micelles loaded with FuOXP alone (179.1 nm) (Figure 2e), suggesting that the siRNA was encapsulated and stabilized in the micelles. Similar results were also shown when siRNA was complexed with drug-free PMBOP micelles.

[0102] Drug carriers with cationic surfaces are not suitable for systemic delivery to distant solid tumors. Therefore, PMBOP / FuOXP / siRNA complexes formed at an N / P ratio of 10 / 1 were subjected to surface coating, application or modification with a mixture / application of chondroitin sulfate (CS) and PEG-CS to form nanoparticles (NPs) of PMBOP-CP. CS is a highly negatively charged molecule and can be used to reduce the positive charge on the surface of the resulting NPs. CS, like hyaluronic acid (HA), is also a natural ligand for CD44, which is overexpressed in various types of cancer cells and tumor endothelial cells (ECs). CS and HA-based NPs have been widely investigated as carriers for tumor targeting. One barrier limiting the efficacy of tumor targeting via HA or CS is the expression of CD44 on liver sinusoidal endothelial cells (LSECs), which, as a result of their abundance, rapidly clear most of the circulating NPs. Indeed, many reported NPs coated with HA and CS showed widespread uptake in the liver, at levels significantly higher than those in the tumor. Therefore, we included a small amount of PEG-CS in the hope of improving the EPR effect by minimizing the “non-specific” uptake by the liver. As shown in Figure 2f, increasing the amount of CS gradually neutralized the positive charge. The NPs obtained with a N / P / sulfate (sulphate:S) ratio of 10 / 1 / 2.25 were 136.5 nm in size and slightly positively charged (+4.2 mV). Under these conditions, the incorporation of small amounts of PEG-CS (CS / PEG-CS molar ratios from 2.5 / 0.1 to 2.25 / 0.25) further neutralized / shielded the positive charges, resulting in near charge neutrality (Figure 2g). Further increase in the amount of PEG-CS led to the formation of negatively charged NPs (Figure 2g).The morphology of siRNA-loaded and unloaded PMBOP-CPNPs was observed by cryo-electron microscopy (cryoEM), revealing spherical particles with relatively uniform diameters in both cases (Fig. 2h). FuOXP loaded in PMBOP-CPNPs showed slow release kinetics in PBS, but the release was accelerated upon exposure to mouse serum (Fig. 2i). When complexed with siRNA, the drug release rate was slightly decreased in both PBS and mouse serum (Fig. 2i). Moreover, siRNA loaded in PMBOP-CPNPs was well protected from degradation by RNase (Fig. 2j).

[0103] In general, any compound with a suitable negative charge can be conjugated with PEG (and / or another hydrophilic polymer) to immobilize the hydrophilic polymer conjugate to the nanostructure. Such compounds can be, for example, other negatively charged CD44 ligands, biological compounds, synthetic compounds, etc. Alternatively, a portion of the hydrophilic polymer can be modified to contain a negative charge.

[0104] For the initial optimization of PMBOP-CP NPs, a sc tumor model (CT26) was used. The in vivo distribution of Cy5.5-labeled siRNA in tumors and liver was examined by fluorescence microscopy 24 hours after iv injection of various PMBOP-CP NPs prepared with an N / P ratio of 10 / 1 and coated with various amounts of CS / PEG-CS, respectively. Increasing the N / P / S (CS) ratio from 10 / 1 / 1 to 10 / 1 / 2.5 gradually increased the Cy5.5 signal in tumors, accompanied by a decrease in the signal in liver. Further increase in the amount of CS decreased the signal in tumors and increased the signal in liver, as measured via fluorescence microscopy (Figure 3a). We next investigated the tumor targeting efficiency of CS-coated NPs and whether the incorporation of PEG-CS could further improve CS-coated NPs. The N / P / S ratio was maintained at 10 / 1 / 2.25 while gradually increasing the amount of PEG-CS. As illustrated in Figure 3B, increasing the CS / PEG-CS ratio from 2.25 / 0.2 to 2.25 / 0.5 further increased the Cy5.5 signal measured via fluorescence microscopy in the tumor but decreased it in the liver. Further increasing the amount of CS-PEG resulted in a decrease in the signal in the tumor and an increase in the signal in the liver (Figure 3b). All subsequent studies were performed with NPs prepared with a N / P / S(CS) / S(PEG-CS) ratio of 10 / 1 / 2.25 / 0.5.

[0105] Figure 3c-C show NIR images at various times after iv injection of Cy5.5-siRNANPs. Whole-body imaging showed that Cy5.5 signals were concentrated in the tumor area at 24 hours (Figure 3c). The ex vivo imaging data (Figure 3d) were consistent with the whole-body imaging results. The level of fluorescent signal in the tumor was significantly higher than that in the liver. Little signal was found in the heart, kidney, spleen and lung. Figure 3e shows that the siRNA signal in the tumor increased with time, peaked at 24 hours and then slowly decreased. siRNANPs remained in the blood significantly longer than free siRNA (Figures 3f and g).

[0106] Effective tumor targeting was also demonstrated in several other sc tumor models, including human colon cancer (WiDr), human breast cancer (BT-474), mouse pancreatic cancer (Panc02), and mouse breast cancer (4T1.2) (Fig. 3h). Similar results were also observed in an orthotopic mouse colon cancer model (Fig. 3i). A potential limitation of NPs is their low efficiency in targeting disseminated tumors and tumor metastases as a result of limited EPR in small tumor lesions. Interestingly, preliminary studies showed that PMBOP-CPNPs effectively accumulated in metastatic tumors in the lungs formed by tail vein injection of CT26 tumor cells, while only minimal signals were observed in the lungs of normal mice (Fig. 3j). Further studies will be performed to clarify the efficiency of tumor targeting at various stages of lung metastasis and the underlying mechanisms. Figure 3k illustrates the widespread distribution of Cy5.5 signals in tumor sections. At higher magnification, the Cy5.5 signal was distributed in both a (predominantly) punctate and diffuse pattern, suggesting that most of the siRNA taken up into the cells was sequestered in endosomes / lysosomes and some was released into the cytoplasm (Fig. 3l-m). Colocalization of Hoechst and Cy5.5 was also observed, suggesting that some siRNA accumulated in the nucleus after escape from endosomes (Fig. 3l and n).

[0107] CD44-mediated transcytoplasmic transport plays a role in tumor targeting. In this regard, the unexpected decrease in tumor uptake associated with increased PEG shielding (PEG-CS / CS > 0.5 / 2.25) suggests that CS-mediated active targeting may play a role in tumor targeting as a whole. To investigate whether CD44-mediated EC targeting plays a role, we used CD44 - / - NIR imaging was also performed in mice. As shown in Figure 4a and Figure 4b, Cy5.5siRNA NPs were highly effective in accumulating in tumor tissues in WT mice. However, CD44 - / - In mice, the Cy5.5 signal in tumor tissue was significantly reduced. - / - In mice, the uptake of Cy5.5 siRNA NPs in the liver was also reduced (Figure 4b and Figure 4d). Interestingly, the Cy5.5 siRNA signal in the blood was significantly reduced by CD44 - / - The increased expression of Cy5.5siRNA NPs in mice (Fig. 4c and Fig. 4e) suggests that Cy5.5siRNA NPs are highly stable in the blood for a significant period of time and that CD44-mediated tumor ECs contribute to tumor-wide targeting, whereas CD44 in LSECs may also contribute to NPs uptake in the liver.

[0108] The efficiency of tumor targeting of PMBOP-CP NPs was also significantly reduced in the zombie mouse model (Figure 4f and Figure 4g). In the zombie mouse model, passive targeting mechanisms such as EPR remain active, but active transendothelial transport is inhibited, suggesting that both active and passive targeting mechanisms contribute to tumor-wide targeting by PMBOP-CP NPs. The zombie mouse mode has been discussed, for example, in Sindhwani, S.et al. The entry of nanoparticles into solid tumors. Nature Materials 19,566-+, doi:10.1038 / s41563-019-0566-2 (2020).

[0109] To further explore the role of CD44 in tumor ECs and LSECs interacting with PMBOP-CPNPs, we investigated the uptake of Cy5.5 siRNA NPs by primary mouse LSECs and human umbilical vein endothelial cells (HUVECs). HUVECs cultured in the absence of basic fibroblast growth factor (bFGF) are quiescent (HUVECq) and express low levels of CD44, whereas HUVECs cultured with bFGF are activated (HUVECa) and express high levels of CD44, which are often used to model tumor ECs. Figure 4h shows that CD44 expression in HUVECa is approximately 1.9- and 13.5-fold higher than that in LSECs and HUVECq, respectively. NPs coated with CS alone (without PEG-CS) were effectively taken up by both HUVECa and LSECs, with more NPs taken up by HUVECa (69.3% vs. 61.5%, p<0.01, Fig. 4i). It was also evident that HUVECq took up significantly less NPs compared to HUVECa (Fig. 4i). Similar results were observed in CD44 cells compared to WTLSECs. - / - This was also demonstrated in LSECs, suggesting that CD44-mediated endocytosis may play a role in the endocytosis of CS-coated NPs by both tumor ECs and LSECs.

[0110] The integration of PEG-CS decreased the cellular uptake of NPs in a PEG dose-dependent manner in both HUVECa and LSECs. However, PEG-CS clearly showed more impact on uptake by LSECs compared to HUVECa. At a CS / PET-CS ratio of 2.25 / 0.25, the uptake level by LSECs decreased to 39.8%, whereas it remained at 58.7% in HUVECa (Figure 4i). Even at a ratio of 2.25 / 0.5, which proved optimal in the in vivo studies herein, a significant difference was observed between the two groups (32.4% vs. 47.6%). At a CS / PEG-CS ratio of 2.5 / 1.0, the uptake level of PMBOP-CPNPs in both cell types decreased to a similar level (24.4% vs. 26.8%). Similar results were obtained when the cellular uptake was evaluated by the mean fluorescence intensity per cell (Figure 4i).

[0111] CD44 has been shown to be capable of mediating transcytosis. To explore the possible role of transcytosis in tumor targeting, a co-culture experiment was performed with HUVECa and CT26 cells using Transwell plates. + As revealed by flow analysis of CT26 cells (Fig. 4j), application of Cy5.5.5-siRNA NPs to HUVECa grown in the upper chamber effectively transduced CT26 cells grown in the lower chamber, which was significantly inhibited by the endocytosisinhibitor Dynasore, suggesting the efficacy of our NPs in mediating transcytoplasmic transport through vascular ECs.

[0112] Of concern is the expression of CD44 on immune cells, such as activated T cells (Ta). However, the CD44 levels on immune cells were significantly lower than those on tumor cells and “tumor ECs” (HUVECa) (Figure 4h). Thus, Cy5.5 +The uptake of NPs by Ta was significantly less than that by tumor cells or tumor ECs, both in terms of the percentage of cells (Fig. 4k) and the mean fluorescence intensity per cell (Fig. 4k). The uptake of NPs by Ta was further reduced in the presence of tumor cells, as in tumor tissues in vivo.

[0113] The above data suggest that the effective tumor targeting of CS-based NPs is “ironically” improved in vivo by shielding with an appropriate amount of PEG (Fig. 4l). CS-NPs without PEG shielding showed limited tumor targeting as a result of the rapid clearance of NPs by LSECs via CD44-mediated uptake. Due to the relatively high levels of CD44 in tumor ECs compared to LSECs, the incorporation of an “optimized” amount of PEG minimizes the interaction of NPs with LSECs without significantly compromising tumor ECs or tumor cell binding when NPs reach tumor cells via EPR (Fig. 4i). Using an excess amount of PEG inhibits the interaction of CS-coated NPs with tumor ECs, resulting in the uptake of NPs by Kupffer cells and possibly LSECs through a CD44-independent mechanism. The slight difference in the optimal ratio in vitro and in vivo may be attributed, among others, to subtle differences in CD44 expression levels between model tumor ECs and mouse tumor ECs.

[0114] The in vivo PK and tissue distribution of siXkr8 and FuOXP after iv administration of FuOXP / siXkr8 NPs, as well as the efficiency of gene knockdown, were also studied. Through the above characterization work, an "optimized" PMBOP-CP nanocarrier with well-defined biophysical properties was developed for the study herein. FuOXP and Cy5.5-siXkr8 were then co-loaded into NPs to study the PK and tissue distribution at different times after iv injection. The kinetics of FuOXP were followed by direct quantification of platinum (Pt) in blood and tissues without extraction using inductively coupled plasma mass spectrometry (ICP-MS). The amount of siRNA was measured by qRT-PCR or fluorimeter after extraction from blood and tissues. Figure 5a shows the Pt concentration in blood over time after iv injection of free FuOXP / siXkr8 or FuOXP / siXkr8NPs into tumor-free mice. Pharmacokinetic parameters were obtained by fitting the blood Pt concentration versus time using a non-compartmental model (Figure 5b). FuOXP formulated in NPs increased t 1 / 2 , AUC and C max It was big about V d and CL were significantly lower than those of free FuOXP. Prolonging the half-life of blood Pt using FuOXPNPs translated into a significant promotion of Pt accumulation in tumor tissue. Pt concentration in tumors increased over time after iv administration. At 24 h after injection, Pt concentration in tumors was about 3.3-fold higher than that in liver (Fig. 5c), and the total amount of Pt in tumor tissues was 12.2% of the injected dose (ID) (Fig. 5d). Pt was mainly detected in the liver and was almost undetectable in tumors 24 h after injection of free FuOXP (Fig. 5c-d). Similar results were observed for the PK and tissue distribution of siXkr8 by either qRT-PCR (Fig. 5e-h) or fluorescence measurement.

[0115] Following the demonstration of enhanced siRNA delivery to tumor tissues via PMBOP-CP NPs, the efficiency of siRNA-mediated knockdown of target genes was further investigated in vitro and in vivo. As shown in Figure 5i, Cy5.5-siRNA NPs were effectively taken up by cultured CT26 cells. Delivery of luciferase siRNA (siLUC) to MC38-Luc cells using PMBOP-CPNPs significantly reduced luciferase activity (Figure 5j). The efficiency of expression suppression was dramatically decreased when cells were pretreated with filipin, chlorpromazine, amiloride, dynasore, cytochalasin D, or MβCD, respectively, prior to gene transfection (Fig. 5j), suggesting that clathrin (CME), caveolin (CvME), flotillin (FME), and Arf6 (ADE)-dependent cellular uptake is involved in siRNA delivery. Fig. 5k shows that Xkr8 mRNA levels were significantly decreased in cultured CT26 cells 20 h after treatment with siXkr8NP.

[0116] In vivo delivery of siLucNPs also resulted in a gradual decrease in luciferase activity in MC38-Luc tumor-bearing mice upon repeated injections, as assessed by whole-body bioluminescence imaging. Tumor luminescence levels were reduced by 34.3% after the first treatment and 80.1% after three treatments (Figure 5l). Figure 5m shows that siXkr8NPs effectively inhibited both basal and FuOXP-delivered Xkr8 mRNA levels in vivo.

[0117] Consistent with literature studies (see W. et al. Synergistic and low adverse effect cancer immunotherapy byimmunogenic chemotherapy and locally expressed PD-L1 trap. Nat Commun 9, 2237,doi:10.1038 / s41467-018-04605-x (2018)), treatment of CT26 tumor cells with FuOXP NPs significantly increased the levels of surface PS (Fig. 6a). This increase was almost abolished when cancer cells were pretreated with siXkr8 and then co-treated with siXkr8. Pretreatment with siXkr8NPs also partially abolished the increase in the amount of FuOXP-transfected PS-positive extracellular vesicles (EVs), which have been reported to be highly immunosuppressive (Fig. 6b).

[0118] Figure 6c shows that co-culture of CT26 cells pretreated with FuOXP / control siRNA (SiCT) NPs with primary mouse macrophages resulted in a decrease in the M1-like / M2-like ratio. These changes were significantly reversed when CT26 cells were first treated with siXkr8 followed by co-treatment with FuOXP / siXkr8.

[0119] The above data prompted us to investigate the therapeutic potential of co-delivery of FuOXP and siXkr8 using PMBOP-CPNPs in the CT26 and Panc02 models. These two models were chosen because 5-FU and oxaliplatin are important therapies for both colon and pancreatic cancer, but have limited efficacy. siXkr8NPs alone only slightly inhibited CT26 tumor growth (Fig. 6d). This result may be due to specific Xkr8 knockdown, since the subline CT26, which shows stable Xkr8 knockdown, showed significant tumor growth retardation in immunocompetent mice. FuOXPNPs alone were only moderately effective in controlling tumor growth (Fig. 6d). However, the combination of both significantly improved antitumor activity (Fig. 6d). Similar results were observed in the Panc02 pancreatic cancer model (Fig. 6e).

[0120] The frequency of PS-positive cells was decreased after treatment with siXkr8NPs alone (Fig. 6f). Treatment with FuOXP NPs dramatically increased the frequency of PS-positive cells, and this increase was almost abolished when FuOXP and siXkr8 were co-delivered into tumors via PMBOP-CPNPs (Fig. 6f).

[0121] Treatment with FuOXP / siXkr8-co-loaded NPs significantly increased CD45 expression compared with the control group or the group treated with FuOXPNPs alone. + cells, IFNγ + CD8 + T cells and GzmB + CD8 + The number of T cells was significantly increased, and CD4 + The number of regulatory T cells was decreased (Fig. 6g-k). Treatment with PMBOP-CP NPs loaded with FuOXP alone or with PMBOP-CP NPs loaded with FuOXP and SiCT significantly decreased the M1-like / M2-like ratio (Fig. 6i). These decreases were almost completely reversed in the group treated with NPs loaded with FuOXP / siXkr8 (Fig. 6i). Similar flow data were observed in the Panc02 tumor model.

[0122] FIG. 6l shows that FuOXP NPs with or without siXkr8 co-loading inhibited CD8 + It was also shown that FuOXP / siXkr8NP caused a significant upregulation of PD-1 expression in T cells, suggesting the possibility of combination therapy with anti-PD-1 antibodies. Indeed, the combination of FuOXP / siXkr8NP with anti-PD-1 resulted in a dramatic improvement in therapeutic efficacy, as evidenced by the significant inhibition of tumor growth (Fig. 6m) and the prolongation of survival time (Fig. 6n).

[0123] FuOXP / siRNA co-loaded NPs were well tolerated at the doses used, as shown by normal weight gain (Fig. 7a), minimal changes in serum levels of ALT and AST (Fig. 9b), and normal histology of several major organs examined (Fig. 7c). Moreover, unlike siRNA complexed with cationic DOTAP liposomes, which caused a significant increase in serum levels of TNF-α and IL-6, siRNAPMBOP-CP NPs did not affect serum levels of the two inflammatory cytokines (Fig. 7d). FuOXP NPs significantly increased the amount of mXkr8 mRNA (Fig. 7e) and PS in the liver. + The number of CD45 cells (Figure 7f) and CD45 expression were minimally affected at the doses used in the therapeutic study. + There was no obvious change in cell number (Fig. 7g).

[0124] In summary, we have shown that Xkr8 expression in tumor cells is significantly triggered by chemotherapeutic agents. Furthermore, our hypothesis-driven study developed a representative PMBOP-CP-based nanocarrier that is highly effective for tumor targeting through effective tumor EC-mediated active targeting while minimizing LSEC-mediated liver uptake. In addition to enhancing the delivery of both types of therapeutics to tumors, our strategy has the advantage of selectively delivering siXkr8 to tumor cells exposed to chemotherapeutic agents. Thus, our co-delivered formulations and methodologies are particularly effective in antagonizing Xkr8 mRNA induced in situ by co-delivered chemotherapeutic agents. Co-delivery of siXkr8 with FuOXP resulted in significant improvement of the tumor immune microenvironment and enhanced antitumor activity. Interestingly, Xkr8 - / - Mice are developmentally normal and do not develop autoimmune disease in C57BL / 6 mice, suggesting that targeting Xkr8 in tumor cells is a safe approach. Safety concerns of targeting Xkr8 can be further minimized by tumor-targeted delivery using NPs. Targeting Xkr8 in combination with chemotherapy may represent a novel and effective immunochemotherapeutic approach for treating various types of cancer, including colon and pancreatic cancer. EXAMPLES

[0125] Experimental Example

[0126] [material] Dulbecco's Modified Eagle's Medium (DMEM), poly(maleic anhydride-alt-1-octadecene) (PMAO, Mn = 30,000-50,000), ethylenediamine, DMF, tert-butanol, and trypsin-EDTA solution were purchased from Sigma-Aldrich (MO, USA). 2K-NHS was purchased from JenKemTechnology (TX, USA). Dicyandiamide was purchased from TCI America (OR, USA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Invitrogen (NY, USA). Antibodies used for flow cytometry were purchased from established sources such as BioLegend and BDBiosciences. Dicyandiamide was purchased from TCI America (PA, USA).

[0127] [Xkr8 siRNA and other oligonucleotides] Mouse Xkr8 siRNA (siXkr8) and control (non-targeting) siRNA (siCT) were designed and synthesized by Ambion (TX, USA). Cy5.5-siXkr8 was synthesized by Sigma-Aldrich (MO, USA). Cy5.5 was introduced into siXkr8 via a phosphate linkage using phosphoramidite chemistry. mXkr8 and hXkr8 mRNAs and primers for RT-PCR amplification of siXkr8 were provided by IDT (IA, USA). The sequences of siXkr8, siCT and other primers are shown in Table 1. All siRNA sequences are non-coding RNAs and were overhanged with deoxythymidine dinucleotides tt and tc at the 3' end to increase the stability of the complex, enhance protection from RNases and provide better gene silencing. The overhangs are not part of the sequence and are not base-paired. In vivo siRNA was quantified using an established method involving two steps, as described, for example, in Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA. 2005Nov;11(11):1737-44. doi: 10.1261 / rna.2148705. PMID: 16244135; PMCID: PMC1370860. In the first step, tailed gene-specific primers (GS primers) were used to convert the RNA template into cDNA. To facilitate subsequent monitoring by qPCR, a "universal" PCR binding site was introduced at one end of the cDNA molecule to extend the length of the cDNA.In the second step, the resulting primer-extended full-length cDNA was quantified by real-time PCR using a combination of a locked nucleic acid (LNA)-containing primer (followed by a +), which restricts the flexibility of the ribofuranose ring and locks the structure into a more rigid two-ring structure, an siRNA-specific "reverse" primer (LNA-R primer), and a general universal primer. [Table 1] RNA: a=adenine, c=cytosine, g=guanine, t=uracil. DNA: a=adenine, c=cytosine, g=guanine, t=thymine. The "+" indicates a restriction / modification in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, "locking" the flexibility of the ribofuranose ring in the nucleobase preceding the "+".

[0128] [mouse] Four- to six-week-old C57BL / 6 mice, BALB / c mice, and NOD.Cg-Prkdc mice were scid Il2rg tm1Wjl / SzJ (NSG) mice and B6.129(Cg)-CD44tm1Hbg / J mice (CD44 - / - Mice) and guinea pigs (Gibco-Myers Squibb) were purchased from The Jackson Laboratories (Connecticut, USA). Mice were housed under pathogen-free conditions in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines. Experiments involving mice were performed in full compliance with institutional guidelines and approved by the Animal Care and Use Advisory Committee at the University of Pittsburgh. Mice were housed at an ambient temperature of 22°C (22°C to 24°C) and 45% humidity, with a 14 / 10 day / night cycle (on at 6:00, off at 20:00) and had free access to food.

[0129] [Tumor cell lines] CT26 and MC38 mouse CRC cell lines, HT29 and WiDr human CRC cell lines, Panc02 mouse PCa cell line, PANC-1 human PCa cell line, 4T1.2 mouse BCa cell line, and BT-474 human BCa cell line were obtained from ATCC (VA, USA). CT26 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin / streptomycin (100 U / mL). MC38 cells, Panc02 cells, PANC-1 cells, 4T1.2 cells, and BT-474 cells were cultured in DMEM medium supplemented with 10% FBS and penicillin / streptomycin (100 U / mL). HT29 cells were cultured in McCoy's 5A medium supplemented with 10% FBS and penicillin / streptomycin (100 U / mL). WiDr cells were cultured in EMEM medium supplemented with 10% FBS and penicillin / streptomycin (100 U / mL). All cells were maintained at 37 °C under 5% CO 2 To establish the mouse tumor model, tumor cells between passages 3 and 10 were cultured in 100 μL of saline at 27 °C. 1 / 2 Inoculations were given subcutaneously (sc) into the right lower quadrant using a G needle.

[0130] [Bulk RNA-seq analysis] RNA-seq was performed by the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh. 3BALB / c mice (n=3) bearing the BRCA1 gene received three iv injections of FuOXPNPs (10 mg FuOXP / kg), once every 5 days, with PBS as a control. Twenty-four hours after the last treatment, tumors were excised. RNA-seq libraries were sequenced as 75-base paired-end reads to a depth of ~73 million to ~77 million reads per sample. Reads were mapped to the mouse genome (GRCm38) using STARAligner 2.6.1a (Dobin, A. et al. STAR: ultrafast universalRNA-seq aligner. Bioinformatics 29, 15-21, doi:10.1093 / bioinformatics / bts635(2013)). Gene expression quantification and differential expression analysis between control and FuOXP NP treatments were performed using Cuffdiff in Cufflinks 2.2.1 (Trapnell, C. et al. Differential analysis of gene regulation attranscript resolution with RNA-seq. Nat Biotechnol 31, 46-53,doi:10.1038 / nbt.2450 (2013)). Volcano plots were created to show overall expression differences, with the x-axis representing the log 2 (fold change)(log 2 FC) and the y-axis shows the corresponding -log 10 (P value) is indicated.

[0131] [Analysis of Xkr8 mRNA levels by qRT-PCR] scCT26 tumor (approximately 200 mm 3 Groups of five BALB / c mice bearing the 14-HT14 receptor agonist (1-HT2) were administered FuOXP NPs, empty NPs or PBS as described above, and tumors were harvested 1 day after the final treatment and subjected to qRT-PCR analysis as detailed below.

[0132] To investigate the effect of various chemotherapeutic agents on Xkr8 mRNA expression, CT26, Panc02, PANC-1, or HT29 cells were treated with various concentrations of FuOXP, DOX, or PTX. After 24 hours, cells were harvested and subjected to qRT-PCR of mXkr8 or hXkr8 as detailed below.

[0133] To examine the effect of ActD treatment on Xkr8 mRNA expression, CT26 or PANC-1 tumor cells were treated with FuOXP (10 μM) for 12 h, followed by the addition of ActD (2 μM). After 2 or 4 h of ActD treatment, cells were harvested and subjected to qRT-PCR of Xkr8 mRNA as described below. In another study, cells were treated with NAC (2.5 mM) for 12 h, followed by treatment with FuOXP, DOX, or PTX for another 12 h. Then, qRT-PCR of Xkr8 mRNA was performed similarly.

[0134] Tumor tissues or cells harvested from the above experiments were incubated with TRIzol TM The purified RNA was subjected to RNA extraction by PCR. cDNA was generated from the purified RNA using the QuantiTect Reverse Transcription Kit (Qiagen, MD, USA) according to the manufacturer's instructions. Quantitative real-time PCR was performed using SYBRGreen Mix in a 7900 HT PCR machine (Applied Biosystems, MA, USA). Relative target mRNA amounts were analyzed using the ΔΔCt method of calculation and normalized to GAPDH. Primer sequences are shown in Table 1.

[0135] [Analysis of hXkr8 protein levels by Western blot] Cultured HT29 or PANC-1 cells were treated in the same manner as above. Then, the cells were lysed using RIPA lysis buffer (ThermoFisher Scientific, Massachusetts, USA) by gentle shaking on ice for 30 min. After centrifugation at 12,300 rpm for 10 min, the supernatant was collected and the protein concentration was measured using PierceBCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). The protein samples were denatured by boiling for 5 min and loaded onto a 10% SDS-PAGE gel for electrophoresis. The proteins in the gel were then transferred onto a PVDF membrane (Bio-Rad, California, USA). The membrane was then incubated in blocking buffer (5% nonfat dry milk in TBST) for 1 h at RT, followed by incubation with anti-hXkr8 polyclonal antibody (ThermoFisher Scientific, MA, USA) in antibody dilution buffer (5% BSA in TBST, 1 / 2000 dilution) overnight at 4°C with gentle agitation. After washing three times with TBST, the membrane was incubated with secondary HRP-conjugated goat anti-rabbit IgG antibody (CellSignaling Technology, MA, USA) for 1 h at RT. After another three washes ... TM The sections were incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA) for 1 min. Protein expression was normalized to β-tubulin expression.

[0136] [Synthesis of PMBOP] Poly(maleic anhydride-alt-1-octadecene) (PMAO, Mn=30,000-50,000, compound 1, 7 g, 20 mmol repeat units) and dry, degassed DMSO (150 mL) were added into a 250 mL glass bottle with a magnetic bar and placed under nitrogen atmosphere. Then, 6.67 mL of ethylenediamine (100 mmol) in 50 mL of dry, degassed DMSO solution was added to the solution. After stirring at 160° C. under nitrogen for 48 hours, the solution was cooled to room temperature and 1 L of HCl solution (2 mol / L) was added. The precipitate was filtered, washed three times with water, and then vacuum dried at 50°C to obtain poly(maleimideethylamine-alt-1-octadecene) polymer (Poly(maleimideethylamine-alt-1-octadecene): PMO, compound 2). Next, 392 mg of compound 2 (1 mmol repeat unit) and 200 mg of PEG were mixed together. 2K-NHS (0.1 mmol), 10 mL of dry DMSO, and 1 mL of TEA (triethylamine) were added into a 50 mL bottle with a magnetic bar. The solution was allowed to stir at room temperature for 48 hours. After the reaction, the solution was transferred to a dialysis bag (MWCO: 12,000-14,000) and dialyzed against water for 24 hours. After dialysis, the solution was filtered through P5 filter paper and lyophilized to obtain PEG-conjugated PMAO polymer with a yield of about 10%-20%. Next, PEG-conjugated PMAO polymer (100 mg) and dicyandiamide (840 mg, 10 mmol) were dissolved in 10 mL of tert-BuOH and refluxed with stirring for 12 hours. After the reaction, the solution was transferred to a dialysis bag (MWCO: 12,000-14,000) and dialyzed against water for 24 hours. After lyophilization, poly(maleimideethylbiscarboximidamide-alt-1-octadecene)-poly(maleimideethylpolyethyleneglycol-alt-1-octadecene) (Poly(maleimideethylbiscarboximidamide-alt-1-octadecene)-Poly(maleimideethyipolyethylene-glycol-alt-1-octadecene: PMBOP, compound 3) was obtained in a yield of approximately 98%.

[0137] [Preparation of PMBOPNPs co-loaded with FuOXP / siRNA] PMBOP polymer (5 mg) and FuOXP (0.5 mg) were dissolved in DMSO in a 10:1 ratio (w / w) and 15 times the initial volume of water was added. The mixture was then purified by HPLC using Amicon (R)The mixture was transferred to an Ultra 3K (MWCO: 3,000) centrifugal filter device (Sigma-Aldrich, MO, USA). After the device was centrifuged at 4,500 rpm for 15 min, 1 mL of water was added. This step was repeated three times to remove residual DMSO, and the FuOXP-loaded micelles were concentrated to the desired volume. After filtering the FITC-labeled PMBOP micelles through a 100 nm pore size membrane, more than 99% of the PMBOP monomers were found to be incorporated into the micelles based on the quantification of free FITC-labeled PMBOP. Next, siXkr8 or siCT (0.1 mg / mL) was mixed with the FuOXP-loaded micelles at an N:P ratio of 10:1 for 20 min at RT to form PMBOP / FuOXP / siXkr8 complexes or PMBOP / FuOXP / siCT complexes. After subsequent incubation with various ratios of CS / PEG-CS mixtures for 20 min at RT, CS / PEG-CS decorated FuOXP / siXkr8 co-loaded PMBOP-CP NPs were formed. CS was found to be quantitatively incorporated (99.12%) into PMBOP-CP NPs under the "optimal" conditions using FITC-labeled CS. Particle size (zeta mean), zeta potential and polydispersity index (PDI) were measured by Zetasizer from three formulation batches. Drug loading capacity (DLC) and drug loading efficiency (DLE) of FuOXP were measured by high-performance liquid chromatography (HPLC). siRNA concentration in the NPs was measured by Ribogreen assay after rupturing the NPs by adding SDS (0.05%), and it was also found that more than 99% of siRNA was incorporated into PMBOP-CP NPs. The complex formation between siRNA and PMBOP polymer was confirmed by gel retardation assay.To examine the resistance of FuOXP / siXkr8 co-loaded NPs to nuclease-mediated degradation, siRNANPs (0.1 mg / mL siRNA, 0.35 mg / mL PMBOP) were incubated with RNase (50 U / mL) (NEB, MA, USA) at 37 °C. After 1 h, the NPs were ruptured with 0.05% SDS and the integrity of the siRNA was examined by electrophoresis. Free siRNA was used as a control. The abbreviation empty NP in this communication refers to PMBOP micelles coated with CS / PEG-CS, and FuOXPNP refers to FuOXP-loaded PMBOP micelles coated with CS / PEG-CS. FuOXP / siRNA NP refers to PMBOP / FuOXP / siRNA complexes coated with CS / PEG-CS.

[0138] [Cryo-electron microscopy] The samples were first checked using negative stain electron microscopy by adding 3 μL to freshly glow-discharged continuous carbon on a copper grid and staining with 1% uranyl acetate solution. The grid was inserted into a ThermoFisher TF20 electron microscope (Thermo Fisher Scientific, MA, USA) equipped with a field emission gun and imaged with a TVIPS XF416 CMOS camera (TVIPS GmbH, Gauting, Germany) to visualize the uniformity and concentration of the nanoparticles. Cryo-grids were prepared by pipetting 3 μL of sample onto Protochips C-flat CF-2 / 1-3CU-T grids (Protochips, NC, USA) that had been glow-discharged for 30 s at 25 mA using an Emitech KX100 glow discharger. The grids were mounted in a Thermofisher Vitrobot Mk 4 at 95% relative humidity, blotted for 3 s at force setting 4, and stained with liquid ethane / propane cooled by a liquid nitrogen bath. 43The grids were transferred onto a Gatan910 three-grid cryoholder (Gatan, Inc., California, USA) and into a TF20 microscope, which was constantly maintained at a temperature below -175°C. The microscope was operated at 200 kV and contrast was enhanced using a 100 μm objective aperture. Cryo-electron micrographs were collected at a nominal 62,000x magnification on a TVIPSXF416 CMOS camera with a 1.3x post-column magnification corresponding to a calibrated pixel size of 1.8 Å at the sample. A low-dose method was used to avoid electron beam damage, and images were acquired with TVIPSEmplified software using movie mode for drift correction. The exposure included a total of 1.5 s exposure with 10 frames every 0.15 s, and the total dose was approximately 10 electrons per square Å.

[0139] [In vitro drug release] The release of FuOXP from FuOXP-loaded PMBOP-CP NPs with or without siRNA complexation was investigated using dialysis. Briefly, 200 μL of FuOXP-loaded PMBOP-CP and FuOXP / siXkr8-loaded PMBOP-CP NPs, each containing 200 μg FuOXP and 2 mg PMBOP, were placed in a dialysis bag (MWCO: 3.5 kDa) containing 5 mL of 0.1 M PBS solution or mouse serum, respectively, and immersed in 40 mL of 0.1 M PBS solution containing 0.5% (w / v) Tween 80. The experiment was carried out in an incubation shaker at 100 rpm at 37°C. At selected time intervals, 10 μL of the solution in the dialysis bag and 1 mL of the medium outside the dialysis bag were removed, and the same amount of new dialysis solution was added for replacement. The concentration of FuOXP was examined by HPLC.

[0140] [Whole-body near-infrared (NIR) fluorescence imaging and ex vivo imaging] Groups of three BALB / c mice were each administered 5 × 10 5CT26 cells were inoculated sc into the right lower abdomen. 3 At the age of 12 years, mice were administered Cy5.5-siXkr8-loaded PMBOP-CPNPs iv at 1 mg / kg siRNA concentration. At 12, 24, and 48 hours, mice were imaged with an IVIS 200 system (Perkin Elmer, Massachusetts, USA) at excitation 679 nm and emission 702 nm with a constant exposure time of 1 second for all groups. After whole-body imaging, mice were euthanized, tumors and various organs were excised, and ex vivo imaging was performed according to published protocols. For blood PK studies, blood was collected into tubes containing Li-heparin at 5 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 48 h, and 72 h, and plasma samples were prepared by centrifugation at 12,300 rpm for 10 min and imaged with the IVIS200 system. Similar studies were also performed in several other sc tumor models, including human colon cancer (WiDr), human breast cancer (BT-474), mouse pancreatic cancer (Panc02), mouse breast cancer (4T1.2, inoculated into the mammary fat pad), and an orthotopic mouse colon cancer model (MC38). To evaluate targeting efficiency in the lung metastasis model, BALB / c mice were inoculated with CT26-luc cells (2 × 10 in 100 μL DPBS) through the tail vein. 5 100 pieces) were injected into the MC38 tumor-bearing mice. 15 days after tumor cell injection, whole-body and ex vivo imaging was performed as described above. As a control, tumor-free mice were injected with NPs loaded with Cy5.5-siRNA. Furthermore, imaging studies were performed on CD44 - / - mice and compared with WT mice.

[0141] [Pharmacokinetics of siXkr8 in blood] Groups of three naive mice received tail vein injections of PMBOP-CP NPs loaded with FuOXP / Cy5.5-mXkr8 siRNA or free FuOXP / Cy5.5-mXkr8 siRNA at doses of 5 mg / kg and 1 mg / kg for FuOXP and siRNA, respectively. For free drug combinations, FuOXP was dissolved in CremophorEL and mixed with siRNA before injection. Blood was collected and plasma prepared at 5 min, 30 min, 1 h, 4 h, and 24 h after injection. The amount of siRNA in the samples was quantified by both qRT-PCR and fluorescence measurement, as detailed below.

[0142] [RT-PCR] siRNA was extracted from samples at various time points using miRNA isolation kit (Invitrogen, CA, USA). SuperScriptIII reverse transcription kit (Invitrogen, MA, USA) was used to convert siRNA to cDNA. For reverse transcription, 6 μL of RT mastermix (2 μL water, 2 μL 5× buffer, 0.5 μL 0.1 M DTT, 0.5 μL 10 mM dNTPs (Invitrogen, MA, USA), 0.5 μL RNaseOUT (Invitrogen, MA, USA) and 0.5 μL SuperScript III enzyme) was mixed with 2 μL of 0.5 μM GS primer and 2 μL of template in a 96-well plate. GS primer and template were premixed and heated at 85° C. for 2 min, then snap-cooled on ice and premixed RT was added. 10 μL of the RT reaction was incubated at 50°C for 30 min, 85°C for 5 min, cooled to room temperature, and then diluted 10-fold with 90 μL of water. After reverse transcription, qPCR was performed in a 384-well optical PCR plate using a 7900HT PCR machine (Applied Biosystems, MA, USA) and 2 μL of cDNA was measured in quadruplicate in a final reaction volume of 10 μL. The SYBRgreen PCR mix contained 5 μL of 2×SYBR green PCR master mix (Applied Biosystems, MA, USA), 1.4 μL of water, 0.8 μL of 10 μM universal primer, 0.8 μL of 10 μM LNA-R primer, and 2 μL of sample. The primer sequences are shown in Table 1. A standard curve was generated by spiking NPs containing Cy5.5-mXkr8siRNA at concentrations ranging from 0 μg / mL to 20 μg / mL into 100 μL aliquots of plasma from untreated animals.

[0143] [Fluorescence measurement] For this assay, an aliquot of plasma (100 μL) was diluted with PBS to a final volume of 500 μL. Methanol (1.0 mL) and chloroform (0.5 mL) were then added, and the sample was vortexed for 2 min until a clear, single-phase solution was obtained. The mixture was then centrifuged at 1,200 rpm for 30 min, and the aqueous phase containing Cy5.5-mXkr8 siRNA was collected. Fluorescence was measured using a Spectramax M5 multiplate reader (Molecular Devices, CA) at an excitation wavelength of 679 nm and an emission wavelength of 702 nm. A calibration curve was similarly constructed as described above.

[0144] [Biodistribution of siXkr8] scCT26 tumor (approximately 300 mm 3 Groups of three BALB / c mice bearing the 5-HT2 gene were injected with PMBOP-CP NPs loaded with FuOXP / Cy5.5-mXkr8 siRNA or free FuOXP / Cy5.5-mXkr8 siRNA via the tail vein at doses of 5 mg / kg and 1 mg / kg for FuOXP and siRNA, respectively. Heart, liver, spleen, lung, kidney and tumor were harvested at 1, 4 and 24 hours and resuspended in 1,000 μL of TRIzol. TM The tissue was homogenized in 5% CO. To the homogenized tissue, 200 μL of chloroform was added. After 5 min of incubation, the samples were centrifuged at 12,300 rpm for 15 min. The upper aqueous phase was used for qRT-PCR and fluorescence quantification as described above. A standard curve was generated by spiking known amounts of Cy5.5-mXkr8 siRNA (0-20 μg / mL) in tissues obtained from untreated control animals and used to calculate the amount of siRNA in the samples.

[0145] [Quantitative analysis of platinum (Pt) by ICP-MS] Plasma and tissue samples were collected as described above. Samples were placed into pre-weighed Purillex PFA bottles (Savillex, MN, USA) and the net weight was recorded. Plasma and tissue samples were frozen at -80°C and lyophilized. 4 mL of HNO 3 Pt (69.0% w / w) and 2 mL of HCl (37% w / w) were added into each PFA bottle, which was then immersed in a water bath at 90°C to digest the samples, resulting in free platinum ions in the lysate. The lysate was then dried at 50°C for 12 hours to remove residual acid. HCl (5%) was added, the samples were transferred into 15 mL centrifuge tubes, and then the Pt concentration in the samples was measured using a PerkinElmer Nexion 300× Inductively Coupled Plasma-Mass Spectrometer (ICP-MS). A calibration curve for Pt in ICP-MS was made by diluting a Pt single element standard (1,000 μg / mL, for AA and ICP, SpexCertiPrep, NJ, USA).

[0146] [Microscopic study of tumor distribution of NPs] For in vivo tumor biodistribution studies, mice bearing CT26 tumors (approximately 300 mm 3 ) were injected i.v. with Cy5.5-siRNA-loaded NPs. Mice were sacrificed 24 h after injection. Frozen sections of tumors were prepared and fixed with acetone at 4°C for 5 min. Cytoskeleton was stained with AF488-Phalloidin (0.33 μM) (CellSignaling Technology, MA, USA) for 15 min at room temperature, and cell nuclei were stained with Hoechst 33324 (1 μg / mL) (ThermoFisher Scientific, MA, USA) for 15 min at room temperature. Tissue sections were then washed three times with cold DPBS before being observed under a confocal laser scanning microscope (CLSM, FluoView1000, Olympus, Japan).

[0147] [Zombie fixation and nanoparticle circulation] NIR imaging in the zombie mouse model was performed according to a previously published protocol (Sun, J.et al. A prodrug micellar carrier assembled from polymers with pendant farnesylthiosalicylic acid moieties for improved delivery of paclitaxel. ActaBiomaterialia 43, 282-291 (2016)). Mice were fixed by transcardial perfusion with 4% formaldehyde and 0.5% glutaraldehyde in PBS for 20 min. Then, perfusion was performed at a physiologically relevant flow rate (6 mL min ) using a peristaltic pump with PMBOP-CPNPs loaded with Cy5.5-siXkr8 at a siRNA concentration of 22.22 μg / mL. -1 ) for 6 hours, after which the mice were imaged with an IVIS 200 system to detect Cy5.5.

[0148] [Uptake into cells] Mouse hepatic sinusoidal endothelial cells (LSECs) were cultured in WT C57BL / 6 mice and in B6.129(Cg)-Cd44 mice. tm1Hbg / J mouse (CD44 - / - mice) from previously published protocols. 47 Briefly, perfused mouse livers were excised from the mice and triturated to release the cells. The cell suspension was then centrifuged several times at different speeds and the suspended pellet was loaded onto the top of a Percoll gradient. Nonparenchymal cells (NPCs) were collected from the interface between two density cushions of 25% with 50% Percoll, and Kupffer cells were removed by selective adherence. LSECs were collected by seeding the cells on collagen-coated cell culture plastic dishes. T cells were isolated from the spleens of naive mice and IL-2 (50 IU / mL) was added every 2 days for 1 week to obtain activated T cells. For cellular uptake studies, WT and CD44 mice were used.- / - LSECs from both mice, mouse (resting and activated) T cells, and subconfluent HUVECs with or without 1% endothelial cell growth supplement (ECGS, ScienCell, CA, USA) treatment (containing growth factors, hormones, and proteins for culturing human capillary endothelial cells) were incubated with Cy5.5-siRNA-loaded PMBOP-CPNPs coated with various amounts of CS / PEG-CS. After 4 hours, cellular uptake was examined by flow cytometry. CD44 expression in LSECs, mouse T cells, and HUVECs with or without growth factor treatment was examined by flow cytometry using antibodies specific for both mouse and human CD44.

[0149] [In vitro gene knockdown] CT26-Luc cells, a CT26 subline stably expressing luciferase, were seeded in antibiotic-free DMEM / FBS in 24-well plates. After 24 hours, the cells were washed with DPBS and incubated in DMEM containing various cellular uptake pathway inhibitors (Table 2) for 1 hour. Then, the cells were treated with PMBOP-CP NPs loaded with luciferase-siRNA (siLuc) at a dose of 100 nM siRNA. Four hours after transfection, the cells were washed with DPBS to remove extracellular siRNA and replaced with DMEM / FBS. 24 hours after transfection, the cells were harvested and subjected to luciferase assay.

[0150] [In vivo gene knockdown] MC38-Luc cells were inoculated sc into the right lower flank of C57BL / 6 mice. PMBOP-CPNP loaded with siLuc or siCT was injected into MC38-Luc tumor-bearing mice at a dose of 2 mg / kg siRNA. Gene knockdown efficiency was measured three times by whole-body bioluminescence imaging on the day after the first, second and third injections, 10, 15 and 20 days after tumor inoculation, respectively. Mice were anesthetized for the first two imaging sessions and euthanized for the final imaging session.

[0151] [Effect of Xkr8 knockdown on PS expression in tumor cells and EVs after FuOXP treatment] CT26 cancer cells were first treated with siXkr8 NPs for 72 h and then with FuOXP / siXkr8 co-loaded NPs for an additional 24 h. EVs were then transfected into the tumor tissue using a previously established protocol. 48 EVs were isolated from the culture medium using gradient ultracentrifugation according to the method described in. Briefly, the culture medium was collected and centrifuged at 1,800 rpm at 4 °C for 20 min to remove dead cells and cell debris. The supernatant was then transferred to a polycarbonate ultracentrifuge tube and centrifuged at 9,000 rpm at 4 °C for 35 min to collect large EVs. The supernatant was centrifuged again at 30,000 rpm at 4 °C for 80 min, the pellet was resuspended in 100 μL of PBS, and small EVs were collected. The amount of surface PS on tumor cells and EVs was analyzed by flow cytometry using Annexin V labeled with BV421. To detect EVs by flow cytometry, the electronic "height" parameter was used instead of the "area" parameter to allow optimal signal detection.

[0152] [Tumor cell / macrophage co-culture study] Mouse macrophages were isolated from the peritoneal cavity according to a previously established protocol. CT26 cancer cells were first treated with siXkr8 NPs for 72 h, then treated with FuOXP / siXkr8 co-loaded NPs for another 24 h in 24-well plates. The treated tumor cells were then transferred and co-cultured with macrophages at a ratio of 10:1 for 72 h, and the macrophages were subjected to flow analysis of M1 / M2 markers, including F4 / 80 (macrophage marker) and CD206 (M2-like macrophage marker). Controls included macrophages not co-cultured with cancer cells, macrophages co-cultured with cancer cells not treated with FuOXP, and macrophages co-cultured with cancer cells treated with FuOXPNPs with or without siCT co-loading.

[0153] [CT26 subline with stable mXkr8 knockdown] A set of three SMARTvector mouse Xkr8 lentiviruses mCMV-TurboGFP shRNA and a SMARTvector control lentivirus mCMV-TurboGFP shRNA were purchased from Horizon Discovery Biosciences (Cambridge, UK). CT26 were transduced in 6-well plates with each lentivirus particle and polybrene at optimal conditions and a given MOI of 10. Cells were incubated for 24 hours, then sorted using TurboGFP and CT26 were transduced. SMARTmXkr8- / - And, CT26 SMART The specimen was collected. CT26 SMART mXkr8- / - , CT26 SMART and CT26 WT were inoculated sc into the right lower abdomen of BALB / c mice, and the tumor size was monitored every 2 days. The tumor volume was calculated by the following formula: tumor volume = 0.5 x length x width. 2 .

[0154] [In vivo therapeutic effect of NPs co-loaded with FuOXP / siXkr8] CT26 or Panc02 tumor cells were inoculated sc into the right lower flank of BALB / c or C57 / BL6 mice. Tumor volumes were approximately 50 mm 3 When tumor size reached 155 mm, mice were randomly assigned to groups (n=5) and intravenously administered DPBS (CT), siCTNP, siXkr8 NP, FuOXP NP, FuOXP / siCT NP, or FuOXP / siXkr8 NP three times at 5-day intervals (days 5, 10, and 15). The doses of FuOXP and siRNA were 5 mg / kg and 1 mg / kg, respectively. Mice were followed up for tumor size and body weight once every 2 days. To verify the therapeutic effect of the combination therapy of FuOXP / siXkr8NP with anti-PD-1, mice were cultured until tumor size reached approximately 155 mm. 3 Treatment was initiated when tumor size reached 100 μg / kg. Mice were treated with anti-PD-1 (clone RMP1-14, BioX Cell, NH, USA) alone, FuOXP / siXkr8 NP alone, or the combination, once every 5 days for three treatments. Anti-PD-1 was administered intraperitoneally (ip) at 200 μg per treatment, and FuOXP / siXkr8NP was administered iv at FuOXP at a dose of 5 mg / kg and siXkr8 at a dose of 1 mg / kg. Mice were followed until death or sacrificed when tumor size reached approximately 2 cm.

[0155] [Analysis of tumor-infiltrating lymphocytes and monocytes] Flow cytometry was performed using LSRII (BD Biosciences, NJ, USA) and Aurora (Cytek Biosciences, CA, USA) instruments and analyzed using FlowJo (BD Biosciences, NJ, USA). Spleens and tumors were harvested 1 day after the final treatment (16 days after the initial tumor inoculation). Single cell suspensions were analyzed using a flow cytometer as previously described. 51Cell cultures were prepared from mouse spleens or tumors as described above. Briefly, tumors were dissected and transferred into RPMI1640. Tumors were mechanically disrupted using scissors, digested with a mixture of DNase I (0.3 mg / mL, Sigma-Aldrich, MO, USA) and Liberase TL (0.25 mg / mL, Roche, Basel, Switzerland) in serum-free RPMI-1640 for 30 min at 37°C, and dispersed through a 40 μm cell strainer (BD Biosciences, NJ, USA). After lysis of red blood cells, live / dead cell discrimination was performed in DPBS at 4°C for 30 min using the ZombieNIR Fixable Viability Kit (BioLegend, CA, USA). Surface staining was performed for 30 min at 4°C in FACS staining buffer (1x phosphate-buffered saline / 5% FBS / 0.5% sodium azide) containing the indicated antibody cocktails (Annexin V, CD45, CD4, CD8, CD11b, Gr-1, F4 / 80, and MHCII). For intracellular protein (Foxp3 and CD206) staining, cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit according to the manufacturer's instructions. For intracellular cytokine (IFN-γ and GzmB) staining, cells were stimulated with phorbol 12-myristate 13-acetate (100 ng / mL) and ionomycin (500 ng / mL) in the presence of monensin for 6 h. Cells were fixed / permeabilized using the BD Cytofix / Cytoperm kit before cell staining.

[0156] [toxicity] The body weight of the mice was monitored once every 2 days during the in vivo therapeutic study. After the end of the experiment, blood samples were collected and ALT and AST were measured by ALT / SGPT or AST / SGOTliqui-UV assay kits according to the manufacturer's protocol. Tumors and major organs including heart, liver, spleen, lungs and kidneys were excised and fixed in 10% formaldehyde in PBS and then embedded in paraffin. Paraffin-embedded samples were cut into thin sections at 4 μm using a HM325 rotary microtome. The tissue sections were then subjected to hematoxylin-eosin staining for histopathological examination under a Zeiss Axiostar plus Microscope (PA, USA).

[0157] In another experiment, naive mice (n=3) received tail vein injection of siRNA complexed with PMBOP-CPNP or DOTAP liposomes loaded with siRNA (N / P:10 / 1) at a dose of 1 mg / kg siRNA. After 2 hours, blood was collected from the orbit and serum cytokine levels (TNF-α and IL-6) were measured using a mouse cytokine assay kit.

[0158] [Statistical analysis] All values ​​were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using two-tailed Student's t-test for comparisons between two groups and one-way analysis of variance (ANOVA) for comparisons between multiple groups. Results were considered statistically significant when p<0.05.

[0159] The foregoing description and accompanying drawings illustrate certain representative embodiments as currently presented. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope of the present specification, which is set forth not by the foregoing description but by the following claims. All changes and variations that come within the meaning and range of equivalency of the claims are embraced within their scope.

Claims

1. a first therapeutic agent for treating the condition; and a second therapeutic agent that can be administered within a predetermined time period of administration of the first therapeutic agent, The second therapeutic agent inhibits the function of Xkr8. Therapeutic systems or combinations.

2. The therapeutic system of claim 1 , wherein the first therapeutic agent induces a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8.

3. The therapeutic system of claim 1 , wherein the second therapeutic agent functions via RNA interference.

4. The therapeutic system of claim 1 , wherein the second therapeutic agent comprises Xkr8 siRNA.

5. The therapeutic system according to claim 1 , wherein the first therapeutic agent is a chemotherapeutic agent.

6. The therapeutic system comprises a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups; a plurality of said first therapeutic agents, said first therapeutic agents being hydrophobic or lipophilic chemotherapeutic agents, are associated with said nanostructure core; The second therapeutic agent, including Xkr8 siRNA, is added to the nanostructure and becomes associated with the cationic groups of the nanostructure. The treatment system according to claim 4.

7. The nanostructure further comprises an application applied to the nanostructure; The application includes a negatively charged targeting agent. The treatment system according to claim 6.

8. The therapeutic system of claim 7 , wherein the negatively charged targeting agent is selected from the group consisting of ligands for cell receptors, peptides, aptamers, polysaccharides and antibodies.

9. The therapeutic system of claim 7 , wherein the negatively charged targeting agent is a ligand for a cell receptor.

10. The treatment system according to claim 8 , wherein the application further comprises a hydrophilic polymer compound.

11. The therapeutic system of claim 10 , wherein the hydrophilic polymer comprises a negative charge.

12. The therapeutic system according to claim 11 , wherein the hydrophilic polymer compound comprises a complex of a negatively charged molecule and a hydrophilic polymer.

13. The therapeutic system of claim 12 , wherein the negatively charged molecule conjugated to the hydrophilic polymer is the same compound as the negatively charged targeting agent.

14. The therapeutic system of claim 9 , wherein the cell receptor is a CD44 ligand.

15. The treatment system according to claim 14 , wherein the application further comprises a hydrophilic polymer compound.

16. The therapeutic system of claim 15 , wherein the hydrophilic polymer comprises a negative charge.

17. The therapeutic system of claim 16 , wherein the hydrophilic polymer compound comprises a complex of a negatively charged molecule and a hydrophilic polymer.

18. The therapeutic system of claim 17 , wherein the negatively charged molecule conjugated to the hydrophilic polymer is a CD44 ligand.

19. The therapeutic system of claim 18, wherein the CD44 ligand is osteopontin, collagen, matrix metalloprotease, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

20. The therapeutic system of claim 18 , wherein the CD44 ligand is chondroitin sulfate.

21. The therapeutic system of claim 15, wherein the second therapeutic agent comprising Xkr8 siRNA is added to the nanostructure before the negatively charged CD44 ligand and the hydrophilic polymer are applied.

22. The therapeutic system of claim 15 , wherein the first therapeutic agent is a small molecule therapeutic compound.

23. 23. The therapeutic system of claim 22, wherein the first therapeutic agent has a molecular weight of less than 1 kDa.

24. The therapeutic system of claim 15 , wherein the cationic groups include groups that are inherently cationic or that form cations in vivo.

25. the group that forms a cation in vivo is an amine group; The amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group.

25. The treatment system of claim 24.

26. 26. The therapeutic system of claim 25, wherein the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

27. 18. The therapeutic system of claim 17, wherein the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides and polypeptides.

28. The therapeutic system of claim 17 , wherein the hydrophilic polymer is polyethylene glycol.

29. The therapeutic system of claim 15, wherein the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is determined so that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of the negatively charged CD44 ligand with CD44 in tumors distant from the liver.

30. 17. The therapeutic system of claim 16, wherein each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one cationic group, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer group.

31. The therapeutic system of claim 30 , wherein the hydrophobic polymer backbone further comprises pendant lipid groups.

32. The therapeutic system of claim 30 , wherein the hydrophobic polymer backbone is formed via free radical polymerization.

33. The therapeutic system of claim 30 , wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

34. administering a first therapeutic agent to treat the condition; administering a second therapeutic agent within a predetermined time period after administering the first therapeutic agent; The second therapeutic agent inhibits the expression level or function of Xkr8. A method of delivering a combination therapy for treating a condition.

35. 35. The method of claim 34, wherein the first therapeutic agent elicits a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8.

36. The method of claim 34 , wherein the second therapeutic agent functions via RNA interference.

37. The method of claim 34, wherein the second therapeutic agent comprises Xkr8 siRNA.

38. 38. The method of any one of claims 34 to 37, wherein the first therapeutic agent is a chemotherapeutic agent.

39. the combination therapy is delivered via a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups; a plurality of said first therapeutic agents, said first therapeutic agents being hydrophobic or lipophilic chemotherapeutic agents, are associated with said nanostructure core; The second therapeutic agent, including Xkr8 siRNA, is added to the nanostructure and becomes associated with the cationic groups of the nanostructure.

38. The method of claim 37.

40. The nanostructure further comprises an application applied to the nanostructure; The application includes a negatively charged targeting agent.

40. The method of claim 39.

41. 41. The method of claim 40, wherein the negatively charged targeting agent is selected from the group consisting of a ligand for a cellular receptor, a peptide, an aptamer, a polysaccharide, and an antibody.

42. 41. The method of claim 40, wherein the negatively charged targeting agent is a ligand for a cell receptor.

43. 42. The method of claim 41, wherein the application further comprises a hydrophilic polymeric compound.

44. The method of claim 43 , wherein the hydrophilic polymer comprises a negative charge.

45. 45. The method of claim 44, wherein the hydrophilic polymer comprises a complex of a negatively charged molecule and a hydrophilic polymer.

46. 46. ​​The method of claim 45, wherein the negatively charged molecule conjugated to the hydrophilic polymer is the same compound as the negatively charged targeting agent.

47. The method of claim 42, wherein the cell receptor is a CD44 ligand.

48. 48. The method of claim 47, wherein the application further comprises a hydrophilic polymeric compound.

49. The method of claim 48 , wherein the hydrophilic polymer comprises a negative charge.

50. 50. The method of claim 49, wherein the hydrophilic polymeric compound comprises a complex of a negatively charged molecule and a hydrophilic polymer.

51. 51. The method of claim 50, wherein the negatively charged molecule conjugated to the hydrophilic polymer is a CD44 ligand.

52. The method of claim 48, wherein the CD44 ligand is osteopontin, collagen, matrix metalloprotease, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

53. a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers; a plurality of first hydrophobic or lipophilic therapeutic agents associated with the core of each of said nanostructures; and a second therapeutic agent that reduces the expression level of Xkr8 or inhibits the function of Xkr8. formulation.

54. the plurality of amphiphilic polymers comprises a cationic group; The second therapeutic agent comprises Xkr8 siRNA that is added to the nanostructure and associates with the cationic groups of the nanostructure.

54. The formulation of claim 53.

55. The nanostructure further comprises an application applied to the nanostructure; The application includes a negatively charged targeting agent.

54. The formulation of claim 53.

56. 56. The formulation of claim 55, wherein the negatively charged targeting agent is selected from the group consisting of a ligand for a cell receptor, a peptide, an aptamer, a polysaccharide, and an antibody.

57. 56. The formulation of claim 55, wherein the negatively charged targeting agent is a ligand for a cell receptor.

58. 57. The formulation of claim 56, wherein the application further comprises a hydrophilic polymer compound.

59. The formulation of claim 58, wherein the hydrophilic polymer comprises a negative charge.

60. 60. The formulation of claim 59, wherein the hydrophilic polymeric compound comprises a complex of a negatively charged molecule and a hydrophilic polymer.

61. 61. The formulation of claim 60, wherein the negatively charged molecule conjugated to the hydrophilic polymer is the same compound as the negatively charged targeting agent.

62. 58. The formulation of claim 57, wherein the cellular receptor is a CD44 ligand.

63. 63. The formulation of claim 62, wherein the application further comprises a hydrophilic polymeric compound.

64. The formulation of claim 63, wherein the hydrophilic polymer comprises a negative charge.

65. The formulation of claim 64, wherein the hydrophilic polymeric compound comprises a complex of a negatively charged molecule and a hydrophilic polymer.

66. 66. The formulation of claim 65, wherein the negatively charged molecule conjugated to the hydrophilic polymer is a CD44 ligand.

67. 64. The formulation of claim 63, wherein the CD44 ligand is osteopontin, collagen, matrix metalloprotease, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

68. 68. The formulation of claim 67, wherein the CD44 ligand is chondroitin sulfate.

69. 64. The formulation of claim 63, wherein the second therapeutic agent comprising Xkr8 siRNA is added to the nanostructure before the negatively charged CD44 ligand and the hydrophilic polymer are applied.

70. 64. The formulation of claim 63, wherein the first hydrophobic or lipophilic therapeutic agent is a small molecule therapeutic compound.

71. 71. The formulation of claim 70, wherein the first hydrophobic or lipophilic therapeutic agent has a molecular weight of less than 1 kDa.

72. 64. The formulation of claim 63, wherein the cationic groups include groups that are inherently cationic or that form cations in vivo.

73. the group that forms a cation in vivo is an amine group; The amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group.

73. The formulation of claim 72.

74. 74. The formulation of claim 73, wherein the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

75. 66. The formulation of claim 65, wherein the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides.

76. 66. The formulation of claim 65, wherein the hydrophilic polymer is polyethylene glycol.

77. The formulation of claim 63, wherein the ratio of the negatively charged CD44 ligand to the hydrophilic polymeric compound is determined so that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of the negatively charged CD44 ligand with CD44 in tumors distant from the liver.

78. 64. The formulation of claim 63, wherein each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one cationic group, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer group.

79. 80. The formulation of claim 78, wherein the hydrophobic polymer backbone further comprises pendant lipid groups.

80. 80. The formulation of claim 79, wherein the hydrophobic polymer backbone is formed via free radical polymerization.

81. 80. The formulation of claim 78, wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

82. administering a first therapeutic agent to treat the condition; administering a second therapeutic agent delivered within a predetermined time from administration of the first therapeutic agent; The second therapeutic agent inhibits the function of Xkr8. How care is delivered to patients.

83. 83. The method of claim 82, wherein the first therapeutic agent elicits a therapeutic response that induces expression of Xkr8 in addition to activating Xkr8.

84. 83. The method of claim 82, wherein the second therapeutic agent functions via RNA interference.

85. 83. The method of claim 82, wherein the second therapeutic agent comprises Xkr8 siRNA.

86. 86. The method of any one of claims 82 to 85, wherein the first therapeutic agent is a chemotherapeutic agent.

87. the first therapeutic agent and the second therapeutic agent are administered via the nanostructure formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups; a plurality of said first therapeutic agents, said first therapeutic agents being hydrophobic or lipophilic chemotherapeutic agents, are associated with said nanostructure core; The second therapeutic agent, including Xkr8 siRNA, is added to the nanostructure and becomes associated with the cationic groups of the nanostructure.

86. The method of claim 85.

88. 86. The method of claim 85, wherein the first therapeutic agent and the second therapeutic agent are delivered in a formulation described in any one of claims 53-81.

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