Biaminoquinolines and nanoformulations for cancer treatment

Abstract

The present invention provides bisaminoquinoline compounds of formula (I). The present invention also provides nanocarriers comprising the compounds of the present invention and methods of using the nanocarriers for disease treatment and imaging.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 902,156, filed September 18, 2019, which is incorporated herein in its entirety for all purposes.

[0002] STATEMENT OF RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant Nos. R01CA199668 and 5R01CA232845 funded by the National Institutes of Health and the National Cancer Institute, Grant No. R01DE029237 funded by the National Institutes of Health and the National Institute of Dental and Craniofacial Institute, and Grant No. R01HD086195 funded by the National Institutes of Health and the National Institute of Child Health and Human Development. The Government has certain rights in this invention. [Background technology]

[0003] Increasing research in nanomedicine has contributed greatly to cancer treatment over the past few decades. Both carrier-assisted and carrier-free nanomedicine are being developed to improve the intrinsic kinetics and safety profile of drugs. However, there are several limitations associated with these nanotherapeutic approaches. First, the complexity and toxicity due to their multicomponent nature have strongly hindered the translation of many nanoformulations to the clinic. Second, most drugs used in traditional delivery studies were approved decades ago, and some are no longer first-line treatments. Third, nanoformulations designed for recently developed therapeutic agents (especially new chemical entities) may encounter patent issues. Finally, not all drugs can be structurally modified. These limitations can be addressed in the context of medicinal chemistry. Compared to nanomedicine, which focuses on delivery profiles for drug research and development, medicinal chemistry focuses on early-stage discovery of drug entities. Although drug discovery techniques have produced a large number of drug leads and candidates, issues surrounding pharmacokinetics, metabolism, and toxicology remain challenges. These challenges may also be relatively easily addressed by nanotechnology from the field of nanomedicine. Leveraging this interdisciplinary link, the principles of nanotechnology to first-line drug design have been integrated to develop a one-component non-prodrug nanomedicine (ONN) strategy ( Figure 1 ), in which drug design follows both traditional drug design strategies and molecular self-assembly principles, thus endowing the designed drugs with advantages from both drug discovery and drug delivery perspectives.

[0004] Lysosomes have been chosen as a target for cancer therapy. Lysosomes in cancer cells are abnormally enlarged and easily disrupted, making them more vulnerable than normal lysosomes. Lysosomal membrane permeabilization (LMP) allows the release of proteolytic enzymes (i.e., cathepsins) into the cytoplasm, which can directly initiate cell death; thus, lysosomotropic detergents capable of inducing LMP have been developed for tumor therapy. Furthermore, lysosomotropic inhibition has considerable potential as an anticancer strategy, since it interferes with autophagy, one important pathway for established tumor stress response and drug resistance. The lysosomotropic alkalinizing agents chloroquine (CQ) and hydroxychloroquine (HCQ) are commonly used autophagy inhibitors and have been tested in numerous clinical trials for various types of cancer. However, their efficacy is considered insufficient, especially when used as single agents.

[0005] Pharmacophore hybridization was employed to design a series of lipophilic cationic BAQ derivatives by molecular self-assembly (Figures 1 and 7). BAQ12 and BAQ13 were selected to construct ONNs because they are potential therapeutic agents and self-assembly building blocks. These BAQ ONNs exhibit excellent anticancer activity in vitro with enhanced effects on lysosomal disruption, lysosomal dysfunction, and autophagy inhibition. Moreover, as nanodrugs, BAQ ONNs exhibit expected self-delivery profiles. These advantages from both drug discovery and drug delivery perspectives ultimately contribute to the remarkable anticancer activity of these compounds as single agents in in vivo gastrointestinal cancer models. In addition, BAQ ONNs are promising for use in combination therapy with napabucasin, since BAQ ONNs play dual roles as both therapeutic agents and delivery carriers. With their multidisciplinary integration and ingenious functional overlap, BAQ ONNs will emerge as an excellent alternative for improving cancer treatment.

[0006] Integrating the unique advantages of the fields of drug discovery and drug delivery is extremely beneficial for the progress of drug development. Herein, we describe a self-delivering, one-component new chemical entity nanomedicine (ONN) strategy to improve cancer therapy through incorporating self-assembly principles into drug design. A lysosomotropic detergent (MSDH) and an autophagy inhibitor (Lys05) are hybridized to develop bisaminoquinoline derivatives that can autonomously form nanoassemblies. The selected ONNs, BAQ12 and BAQ13, are highly effective in inducing lysosomal disruption, lysosomal dysfunction, and autophagy blockade, and exhibit 30-fold greater antiproliferative activity than hydroxychloroquine used in clinical trials. These single-drug nanoparticles exhibit excellent pharmacokinetic and toxicological profiles and dramatic in vivo antitumor efficacy. In addition, these single-drug nanoparticles can encapsulate and deliver additional drugs to tumor sites, making them promising agents for combination therapy based on autophagy inhibition. Given their cross-cutting advantages, these BAQ ONNs have enormous potential to improve cancer therapy. What is needed are new BAQ ONNs. Surprisingly, the present invention meets this need and others. Summary of the Invention

[0007] In one embodiment, the present invention provides a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c W is substituted with C 3-12 Cycloalkyl, C6-12 aryl, or 5-12 membered heteroaryl having 1-4 heteroatoms, each of which is independently N, O, or S, wherein each cycloalkyl, aryl, and heteroaryl is optionally selected from the group consisting of C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 alkynyl; each L is independently absent, C 1-20 Alkylene, C 2-20 Alkenylene, or C 2-20 each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic agent, a sterol, C 3-12 cycloalkyl, 3-12 membered heterocycloalkyl having 1-4 heteroatoms, each independently being N, O, or S; C 6-12 aryl, 5-12 membered heteroaryl having 1-4 heteroatoms, each of which is independently N, O, or S, -OH, or -NH; R 1a is C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-40 Alkoxy, hydroxyl, or -NR 1b R 1c is substituted with;R 1b is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 Alkynyl; R 1c is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 alkynyl, or -LW; R 2a , R 2b , R 3a , and R 3b are each independently hydrogen, C 1-40 Alkyl, C 2-40Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, halogen, -CN, or -NO2; R 2a , R 2b , R 3a , and R 3b are each independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 alkoxy, halogen, -CN, or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; each X is independently absent or -O - However, X is absent and R 2a and R 2b is hydrogen, R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, boron, or -NO2, R 1 is C 2-40 Alkyl, C 2-40 Alkenyl, C 4-40 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a , X is absent, and R 1 is -CH2CH2NH(7-chloro-4-quinolinyl), R 2a and R 2b When is hydrogen, R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 alkoxy, fluorine, boron, iodine, -CN, or -NO2).

[0008] In another embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds of the present invention, or pharma- ceutically acceptable salts thereof, wherein each compound self-assembles in an aqueous solvent to form the nanocarrier, such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0009] In another embodiment, the present invention provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the present invention.

[0010] In another embodiment, the present invention provides a method of imaging comprising administering to a subject to be imaged an effective amount of a nanocarrier of the present invention. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows the proposed drug design strategy and a schematic diagram of this study. Pathway (a) shows that we propose a multidisciplinary drug design strategy to integrate the fields of traditional medicinal chemistry and nanomedicine. The drugs, named one-component non-prodrug nanomedicines (ONNs), will be designed following the strategies of traditional drug design and molecular self-assembly, thus gaining advantages from both drug discovery and drug delivery perspectives. Pathway (b) shows the proof-of-concept experiment in this study, i.e., the discovery of self-delivering lysosomotropic bisaminoquinoline (BAQ) derivatives for cancer therapy. The BAQ derivatives are generated from the hybridization of lysosomotropic detergents and BAQ-based autophagy inhibitors and can self-assemble into BAQ ONNs. As a single agent, the BAQ ONNs exhibit enhanced function in vitro, excellent delivery profile, and remarkable therapeutic efficacy in vivo. Moreover, the BAQ ONNs also possess large drug-loading capacity to deliver additional drugs to tumor sites, thus generating promising applications in combination therapy.

[0012] [Figure 2A-2C]Figures 2A-2G show the characterization of BAQ ONNs. Figure 2A shows the size change of BAQ NPs (10 μM) in acetate buffers with different pH values; data are means ± SD; n = 3 independent nanoparticle samples. Figure 2B shows the pH-dependent hemolysis induced by BAQ NPs (50 μM, 4 h) in PBS buffer; data are means ± SD; n = 3 independent nanoparticle samples. Figure 2C shows the pH change of BAQ NPs (1 mM) over the range of hydrochloric acid (HCl, 0.1 M) titration. [Fig. 2D-2G] Figures 2A-2G show the characterization of BAQ ONNs. Figures 2D-2E show representative TEM microscopy images at pH 7.4 (Figure 2D) and pH 5.0 (Figure 2E); insets show size distribution (left) and Tyndall effect (right); all experiments were repeated three times independently. Figure 2F shows the in vitro drug release patterns at pH 7.4 and pH 5.0; data are means ± SD; n = 3 independent nanoparticle samples. Figure 2G shows the count rates of BAQ NPs at various concentrations in water; the intersection of the two lines is the CAC of BAQ12 NPs (0.45 μg mL-1, 0.76 μM) and BAQ13 NPs (0.15 μg mL-1, 0.25 μM); data are means ± SD; n = 3 independent nanoparticle samples.

[0013] [Figure 3A] Figures 3A-3K show lysosomal disruption and inhibited autophagy induced by BAQ ONN in MIA PaCa-2 cells. Figure 3A shows representative images of nanoparticle uptake into cells; Dextran-AF488-loaded cells were incubated with DiD-labeled BAQ ONN for 2 h; the experiment was repeated three times independently. [Figure 3B-3D]Figures 3A-3K show lysosomal disruption and inhibited autophagy induced by BAQ ONN in MIA PaCa-2 cells. Figure 3B shows cells treated as indicated (10 μM, 2 h) and stained with LysoTracker Green; experiments were repeated three times independently. Figure 3C shows AO staining of cells at the range of treatments indicated (5 μM, 12 h); experiments were repeated three times independently. Figure 3D shows representative images of cells loaded with Dextran-AF488 treated as indicated (5 μM, 12 h); experiments were repeated three times independently. [Figure 3E-3G] Figures 3A-3K show lysosomal disruption and inhibited autophagy induced by BAQ ONN in MIA PaCa-2 cells. Figure 3E shows cathepsin B release from isolated lysosomes after treatment as indicated (25 μM, 12 h); data are shown as mean ± SD; n = 3. Figure 3F shows Western blotting. Figure 3G shows quantitative analysis of normalized gel blots in Figure 3F; data are shown as mean ± SD; n = 3. [Figure 3H-3K] Figures 3A-3K show lysosomal disruption and inhibited autophagy induced by BAQ ONN in MIA PaCa-2 cells. Figure 3H shows representative LC3B-GFP images for 4 h treatment as indicated; experiments were repeated three times independently. Figure 3I shows quantification of LC3B-GFP puncta per cell at h; data shown as mean ± SD; n = 3. Figure 3J shows representative TEM images of cells treated as indicated (2 μM, 48 h); orange rectangle: region of interest; purple arrow: autophagic vesicle; red arrow: lysosome. Figure 3K shows the average diameter of lysosomes; data shown as mean ± SD; n = 7. All p values ​​of statistics were calculated by one-way ANOVA with Tukey's multiple comparison test. ns., not significant; *p<0.05; **p<0.01; ****p<0.0001.

[0014] [Figure 4A-4C]Figures 4A-4J show that BAQ ONN altered the expression of lysosomal genes and caused cell death through apoptosis. Figure 4A shows GSEA demonstrating enrichment of lysosomal gene sets in MIA PaCa-2 cells treated with BAQ13 NPs (5 μM, 24 h). GSEA was performed with n=1,000 permutations and adjusted p<0.05 and FDR<0.05 were considered significant. Figure 4B shows representative upregulated lysosomal genes from a. Figure 4C shows a comparison of gene upregulation between Lys05 and BAQ13 NPs.

[0015] [Fig. 4D-4G] Figures 4A-4J show that BAQ ONN altered the expression of lysosomal genes and caused cell death through apoptosis. Figures 4D-4E show qPCR analysis of V-ATPase genes (Figure 4D) and Cl- channel genes (Figure 4E) involved in the indicated treatments (5 μM, 24 h); data are means ± SD; n = 3. Figure 4F shows the survival curves of cells subjected to 48 h of treatment and the corresponding IC50 values; data are means ± SD; n = 3. Figure 4G shows the proliferation curves of MIA PaCa-2 (1.5 μM) and HT29 (1.0 μM) cells over a range of continuous treatments; data are means ± SD; n = 3 independent experiments. [Fig. 4H-4J] Figures 4A-4J show that BAQ ONN altered the expression of lysosomal genes and caused cell death through apoptosis. Figure 4H shows the clonogenic assay of MIA PaCa-2 cells and HT29 cells; n = 3. Figure 4I shows the activity of caspase 3 / 7 in MIA PaCa-2 cells and HT29 cells treated for 6 and 12 h, respectively; data are mean ± SD; n = 4. Figure 4J shows the percentage of apoptotic population in MIA PaCa-2 cells (left) and HT29 cells (right) treated for 24 h. All p values ​​of statistics were calculated by one-way ANOVA with Tukey's multiple comparison test; ns., not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0016] [Figure 5A-5C] Figures 5A-5H show the pharmacokinetics, biodistribution, and in vivo antitumor effects of BAQ ONNs. Figure 5A shows the plasma concentration-time profiles after intravenous injection of DiD-loaded BAQ ONNs and free DiD; data are means ± SD; n = 3. Figure 5B shows the in vivo and in vitro distribution 24 h after injection of BAQ13 NPs in HT29 tumor-bearing mice. Figure 5C shows the quantitative fluorescence intensity of tissues obtained at 12 and 24 h post-injection; data are means ± SD; n = 3. [Fig. 5D-5G] Figures 5A-5H show the pharmacokinetics, biodistribution, and in vivo antitumor effects of BAQ ONN. Figure 5D shows MIA PaCa-2 tumor growth curves in mice treated every 3 days as indicated; data are mean ± SD; n = 6 tumors per group. Figure 5E shows the body weights of mice during treatment; data are mean ± SD; n = 6 mice per group. Figure 5F shows the weights of tumors harvested at the end of treatment; data are mean ± SD; n = 6 tumors per group. (g-j) Figures 5G-J show representative H&E (Figure 5G), IHC (Figure 5H), immunoblotting (Figure 5I), and TEM (Figure 5J) results of tumors harvested at the end of treatment; i blots for each group were derived from three individual tumors in each group; purple arrows in Figure 5J: autophagic vesicles; all experiments in Figures 5G-5J were independently repeated three times. All statistical p-values ​​were calculated by one-way ANOVA with Tukey's multiple comparison test; *p<0.05; ****p<0.0001. [Figure 5H] (g-j) Figure 5G-J show representative H&E (Figure 5G), IHC (Figure 5H), immunoblotting (Figure 5I), and TEM (Figure 5J) results of tumors harvested at the end of treatment; i blots for each group were derived from three individual tumors in each group; purple arrows in Figure 5J: autophagic vesicles; all experiments in Figure 5G-5J were independently repeated three times. All p values ​​of statistics were calculated by one-way ANOVA with Tukey's multiple comparison test; *p<0.05; ****p<0.0001.

[0017] [Figure 6A-6B] Figures 6A-6L show that BAQ ONN has a dual role in combination therapy. Figure 6A shows the establishment of a patient-derived pancreatic cancer stem cell (PCSC) model. Figure 6B shows histological analysis showing high levels of stroma in PCSC tumors; experiments were repeated three times independently. [Figure 6C-6F] Figures 6A-6L show that BAQ ONN has a dual role in combination therapy. Figure 6C shows the survival curve and IC50 value of PCSCs treated for 48 h; n = 3 independent experiments. Figure 6D shows AO staining showing LMPs of PCSCs treated for 12 h; experiment was repeated three times independently. Figure 6E shows immunoblotting analysis of autophagy proteins in PCSCs treated as indicated (2.5 μM, 48 h); experiment was repeated three times independently. Figure 6F shows the synergistic effect of BAQ13 NPs and napabucasin (48 h). [Figure 6G-6I] Figures 6A-6L show that BAQ ONN has a dual role in combination therapy. Figure 3G shows tumor growth curves in a subcutaneous MIA PaCa-2 xenograft model over a range of intravenous administration every 3 days; data are means ± SD; n = 10 tumors per group. Figure 3H shows images of tumors harvested at the end of treatment. Figure 3I shows the weight change of mice during treatment; data are means ± SD; n = 5 mice per group. [Fig. 6K-6L] Figures 6A-6L show that BAQ ONNs have a dual role in combination therapy. Figure 3J shows representative images of PCSC tumor sections; the experiment was independently repeated three times. Figure 3K shows in vivo and ex vivo fluorescence imaging of BAQ13 NPs co-loaded with napabucasin and DiD in a PCSC model 48 h after intravenous injection (10 mg kg-1). Figure 3L shows the quantitative fluorescence intensity of tissues in Figure 3K, data are mean ± SD; n = 3 mice per group. All p values ​​of statistics were calculated by two-tailed Student's t-test. ns., not significant; *p<0.05; **p<0.01; ****p<0.0001.

[0018] [Figure 7] Figure 7 shows the chemical structures of the compounds involved in this study and the synthetic routes of BAQ12 to BAQ18.

[0019] [Figure 8A-8B] Figures 8A-8C show the in vitro evaluation of BAQ NPs. Figure 8A shows the size distribution. Figure 8B shows the observation of a pH-dependent hemolytic effect; red blood cells were treated as indicated (50 μM, 4 h). [Figure 8C] Figures 8A-8C show the in vitro evaluation of BAQ NPs. Figure 8C shows the survival curves of various cell lines exposed to different compounds for 24 hours. Data are presented as mean ± SD; n = 3 independent experiments.

[0020] [Figure 9A-9C] Figures 9A-9G show the stability measurements and TEM characterization of BAQ NPs. Figures 9A-9B show the dynamic light scattering (DLS) measurements of BAQ12 NPs and BAQ13 NPs at neutral conditions; data are shown as mean ± SD; n=3. [Fig. 9D-9G] Figures 9A-9G show the stability measurements and TEM characterization of BAQ NPs. Figure 9C shows the overall appearance of BAQ12 NPs and BAQ13 NPs at 10 and 30 days. Figures 9D-9E show the DLS measurements of BAQ12 NPs and BAQ13 NPs in the presence of 10% FBS; data are shown as mean ± SD; n = 3. Figure 9F shows the size distribution of BAQ12 NPs (left) and BAQ13 NPs (right) upon incubation with 0.5 mM BSA for 24 h. Figure 9G shows representative TEM images of BAQ13 NPs loaded with different drugs; the experiment was repeated three times independently.

[0021] [Figure 10]Figures 10A-10F show the effect of BAQ NPs on lysosomes and autophagy at the cellular level. Figure 10A shows the Pearson correlation coefficient for the colocalization analysis of Figure 3A. Figure 10B shows the fluorescence quantification of LysoTracker Green in MIA PaCa-2 cells treated as indicated; data are shown as mean ± SD; n = 3 independent experiments; statistical significance was calculated by two-tailed Student's t-test; **p < 0.01. Figure 10C shows HT29 cells treated as indicated (10 μM, 2 h) and then stained with LysoTracker Red; experiments were repeated three times independently. Figure 10D shows AO staining of HT29 cells treated as indicated (5 μM, 12 h); experiments were repeated three times independently. Figure 10E shows the ratio of fluorescence intensity at 525 nm to 650 nm in MIA PaCa-2 cells treated for 12 h as indicated; data are shown as mean ± SD; n = 3 independent experiments; statistical significance was calculated by one-way ANOVA with Tukey's multiple comparison test; ns., not significant; ****p < 0.0001. Figure 10F shows representative LC3B-GFP images of cells with a range of corresponding treatments (5 μM, 4 h); experiments were repeated three times independently.

[0022] [Figures 11A-11D]Figures 11A-11F show the effects of BAQ NPs on gene expression and lipid metabolism. Figure 11A shows volcano plots from RNA-seq showing differentially expressed genes in MIA PaCa-2 cells induced by Lys05 (bottom) and BAQ13 NPs (top). Figure 11B shows changes in autophagy-related genes from RNA-seq results. Figure 11C shows qPCR analysis of representative autophagy- and apoptosis-related genes in MIA PaCa-2 cells treated as indicated (5 μM, 24 h); data are shown as mean ± SD; n=3; statistical significance was calculated by one-way ANOVA with Tukey's multiple comparison test; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. FIG. 11D shows the changes in apoptosis-related genes in MIA PaCa-2 cells treated as indicated (5 μM, 12 h); data are shown as mean ± SD; n = 3; statistical significance was calculated by two-tailed Student's t test; *p < 0.05; **p < 0.01. [Fig. 11E-11F] Figures 11A-11F show the effects of BAQ NPs on gene expression and lipid metabolism. Figures 11E-11F show the concentration changes of acid sphingomyelinase (ASM) substrate (Figure 11E) and phospholipase A (PLA) substrate (Figure 11F) in MIA PaCa-2 cells with or without treatment (2.5 μM, 48 h); data are shown as mean ± SD; n=4.

[0023] [Figure 12] Figure 12 shows the survival curves of various cell lines treated as indicated for 48 hours. Data are presented as mean ± SD; n = 3 independent experiments.

[0024] [Figures 13A-13C]Figures 13A-13F show biodistribution and toxicity studies of BAQ NPs. Figure 13A shows in vivo and ex vivo imaging of HT29 tumor-bearing mice 12 hours after (iv) injection of free DiD (top) and DiD-loaded BAQ13 NPs (bottom), n=3 mice per group. Figure 13B shows quantitative fluorescence intensity of organs in a; data presented as mean ± SD; n=3 mice per group; data presented as mean ± SD; n=3; statistical significance calculated by two-tailed Student's t-test; ***p<0.001; ****p<0.0001. FIG. 13C shows concentration-dependent hemolysis induced by corresponding treatments at physiological pH; data are presented as mean ± SD; n = 3 independent nanoparticle samples; statistical significance was calculated by one-way ANOVA with Tukey's multiple comparison test; ns., not significant; ***p < 0.001; ****p < 0.0001. [Fig. 13D-13F] Figures 13A-13F show biodistribution and toxicity studies of BAQ NPs. Figure 13D shows the survival rate of FVB / n mice injected iv with the corresponding drugs every 2 days; n = 6 mice per group. Figure 13E shows the weight of mice treated every 2 days as indicated; data are presented as mean ± SD; n = 4 mice per group. Figure 13F shows dynamic light scattering (DLS) measurements and representative TEM images of liposomes@Lys05; experiments were repeated three times independently.

[0025] [Figure 14]Figures 14A-14E show histological sections and hematology analysis. Figure 14A shows H&E analysis of major organs from mice treated with vehicle (saline), Lys05 (20 mg kg-1, ip), BAQ12 NPs (20 mg kg-1, iv), and BAQ13 NPs (20 mg kg-1, iv) for 24 days. Scale bars are 100 μm. Experiments were repeated three times independently. Figure 14B shows the corresponding serum chemistry analysis of mice in a. 1. Alanine transaminase U L-1, 2. Aspartate transaminase U L-1, 3. Serum urea nitrogen mg dL-1, 4. Creatinine mg dL-1, 5. Total bilirubin mg dL-1, 6. Hemolysis. 14B-14E show the corresponding complete blood count (CBC) analyses of the mice in a; data are presented as mean ± SD; n=3 mice per group. 1. Absolute number of neutrophil cells (k μL-1), 2. Absolute number of monocytes (k μL-1), 3. Absolute number of eosinophil cells (k μL-1), 4. Absolute number of basophil cells (k μL-1), 5. Eosinophil %, 6. Basophil %, 7. MPV(fL), 8. Absolute lymphocyte cell number (k μL-1), 9. WBC(k μL-1), 10. RBC (M μL-1), 11. Hemoglobin (g dL-1), 12. MCH (pg), 13. Monocytes (%), 14. RDW (%), 15. Neutrophils %, 16. Lymphocytes %, 17. Hematocrit %, 18. MCV (fL), 19. MCHC (g dL-1), 20. Platelets (k μL-1), 21. Presence of blood clots.

[0026] [Figure 15]Figures 15A-15C show the in vivo evaluation of BAQ NPs in a HT29 mouse model. Figure 15A shows the tumor growth curve in a subcutaneous HT29 xenograft model over the range of intravenous administration every 2 days; data shown as mean ± SD; n=12 tumors per group. Statistical significance was calculated by two-tailed Student's t-test; ns., not significant; *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. Figure 15B shows the body weight of mice during treatment; data shown as mean ± SD; n=6 mice per group. Figure 15C shows the survival curve of mice; n=6 mice per group. Treatment was terminated on day 24.

[0027] [Figure 16] FIG. 16 shows the gating strategy for isolating pancreatic cancer stem cells (PCSCs).

[0028] [Figures 17A-17D] Figures 17A-17F show the evaluation of BAQ NPs in a model of pancreatic cancer stem cells (PCSCs). Figure 17A shows a lysosomal deoxidation assay. PCSCs were treated for 2 hours (10 μM) and then stained with LysoTracker Red; the experiment was repeated independently three times. Figure 17B shows representative images of PCSCs treated as indicated (5 μM, 4 hours) and expressing LC3B-GFP; the experiment was repeated independently three times. Figure 17C shows the percentage of apoptotic population of PCSCs at the range of treatments indicated (5 μM, 24 hours). Figure 17D shows an immunoblotting assay of PCSCs treated for 8 hours as indicated; the experiment was repeated independently three times. [Fig. 17E-17F] Figures 17A-17F show the evaluation of BAQ NPs in a model of pancreatic cancer stem cells (PCSCs). Figure 17E shows the H&E analysis of major organs harvested at the end of treatment; the experiment was repeated three times independently. Figure 17F shows the biodistribution of free DiD and BAQ13 NPs@napabucasin+DiD in mice 24 hours after tail vein injection; red circle: tumor.

[0029] [Figures 18A-18D] Figures 18A-18G show the characterization of PBC NPs at physiological and lysosomal pH. Figure 18A shows the chemical structure of the PBC monomer. Figure 18B shows a schematic diagram of the deformability at different pH. Figure 18C shows the ultrafiltration analysis and TEM observation of PBC NPs at pH 7.4 and pH 5.0. Figure 18D shows the pH-dependent DLS measurements of PBC nanoparticles. [Fig. 18E-18G] Figures 18A-18G show the characterization of PBC NPs at physiological pH and lysosomal pH. Figure 18E shows time-dependent DLS measurements of PBC nanoparticles at pH 5.0. Figure 18F shows pH-dependent TEM measurements of PBC nanoparticles. Figure 18G shows time-dependent TEM measurements of PBC nanoparticles.

[0030] [Figure 19A] Figures 19A-19H show that the deformability of PBC nanoparticles induces lysosomal dysfunction and causes apoptosis in OSC-3 cells. Figure 19A shows the chemical structures of deformable PBC nanoparticles and non-deformable BAQ nanoparticles. [Fig. 19B-19D] Figures 19A-19H show that the transformability of PBC nanoparticles induces lysosomal dysfunction and causes apoptosis in OSC-3 cells. Figure 19B shows cell viability, Figure 19C shows autophagy analysis, and Figure 19D shows apoptosis analysis. [Fig. 19E-19H] Figures 19A-19H show that the deformability of PBC nanoparticles induces lysosomal dysfunction and causes apoptosis in OSC-3 cells. Figure 19E shows TEM images demonstrating PBC nanofibers formed in lysosomes. Figure 19F shows acridine orange (AO) staining to show lysosomal membranes (LMPs) induced by PBC NPs. Figure 19G shows dextran staining to show LMPs induced by PBC NPs. (h) PBC NPs induced obvious vacuole formation in OSC-3 cells.

[0031] [Fig. 20A-20E]Figures 20A-20L show that PBC NPs exhibited significant photodynamic effects and could overcome autophagy-associated drug resistance. Figures 20A-20B show that the traditional photosensitizer pheophorbide a (Pa) induced autophagy in OSC-3 cells, which was confirmed by LC3-GFP-RFP imaging (Figure 20A) and immunoblotting (Figure 20B). Figures 20C-20D show that Pa-mediated photodynamic therapy was sensitized by the autophagy inhibitor Lys05, which was confirmed by cell viability (Figure 20C) and immunoblotting (Figure 20D). Figure 20E shows the generation of singlet oxygen. [Fig. 20F-20J] Figures 20A-20L show that PBC NPs exhibited significant photodynamic effects and could overcome autophagy-related drug resistance. Figure 20F shows ROS generation in OSC-3 cells. Figure 20G shows live / dead staining with DiO / PI of OSC-3 oral cells after different treatments. Figure 20H shows cell viability. Figure 20I shows apoptosis assay. Figure 20J shows immunoblotting assay for apoptosis pathway. [Fig. 20K-20L] Figures 20A-20L show that PBC NPs exhibited significant photodynamic effects and could overcome autophagy-related drug resistance. Figure 20K shows TEM images of OSC-3 cells after different treatments. Figure 20L shows immunoblotting assay for the autophagy process.

[0032] [Figure 21] FIG. 21 shows other synthesized BAQ derivatives and their cell viability results (48 hours) in OSC-3 cells.

[0033] [Figure 22] Figures 22A-22B show preliminary screening of BAQO derivatives. Figure 22A shows the chemical structures. Figure 22B shows the results of viability of pancreatic cancer stem cells (PCSCs) treated for 72 hours.

[0034] [Figure 23]Figures 23A-23F show the characterization of BAQ12O NPs. Figure 23A shows DLS measurements of BAQ12O NPs. Figure 23B shows a representative TEM image of BAQ12O NPs. Figure 23C shows the measurement of the critical aggregation concentration (CAC) of BAQ12O NPs. Figures 23D-23E show the absorption (Figure 23D) and fluorescence (Figure 23E) spectra of BAQ12O NPs and free BAQ12O solutions. Figure 23F Stability of BAQ12O NPs.

[0035] [Figure 24] Figures 24A-24F show the evaluation of antitumor activity of BAQ12O NPs in mice bearing PCSC tumors. Figure 24A shows the tumor growth curves in mice treated every 3 days (iv). Figure 24B Tumor weights harvested at the end of treatment. Figure 24C Mouse body weights during treatment. Figure 24D Immunoblotting of autophagy process in mice. Figures 24E-24F show representative HE (Figure 24E) and Ki67-IHC (Figure 24F) images of tumors in different groups.

[0036] [Diagram 25] FIG. 25 shows the chemical structures of the designed BAQO derivatives. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] I. General

[0038] The present invention provides bisaminoquinoline derivative compounds and nanocarriers formed from these compounds that are useful for treating diseases. These compounds and nanocarriers can target lysosomes, resulting in lysosomal disruption, lysosomal dysfunction, and / or autophagy inhibition. Furthermore, the nanocarriers can be used in combination therapy by encapsulating additional drugs or for co-administration with additional drugs that may help overcome drug resistance. The nanocarriers can also be used for imaging of cells or organisms of interest. II. Definition

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, the present invention can be practiced using methods or materials similar or equivalent to any methods or materials described herein. For purposes of the present invention, the following terms are defined:

[0040] As used herein, "a" or "the" includes aspects having one member as well as aspects having two or more members. For example, the singular forms "a" and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the agent" includes one or more agents known to those of skill in the art, and so forth.

[0041] "Alkyl" means a linear or branched saturated aliphatic group having the indicated number of carbon atoms. Alkyl can contain any number of carbons (C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 1-20 , C 1-30 , C 1-40 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C 3-5 , C 3-6 , C 4-5 , C 4-6 , and C 5-6 For example, C 1-6Non-limiting examples of alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 40 carbon atoms, non-limiting examples of which are heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted.

[0042] "Alkylene" means a linear or branched saturated aliphatic group having the indicated number of carbon atoms and linking at least two other groups, i.e., a divalent hydrocarbon group. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For example, as in a straight chain alkylene, -(CH2) n -divalent radicals are possible, where n is 1, 2, 3, 4, 5, or 6. Non-limiting examples of representative alkylene groups include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene. Alkylene groups can be substituted or unsubstituted.

[0043] "Alkenyl" means a straight or branched hydrocarbon having at least two carbon atoms and at least one double bond. Alkenyl can contain any number of carbons (C, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C 2-20 , C 2-30 , C 2-40 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6, and C6). Alkenyl groups can have any suitable number of double bonds, non-limiting examples of which include 1, 2, 3, 4, 5, or more. Non-limiting examples of alkenyl groups include vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substituted or unsubstituted.

[0044] "Alkenylene" refers to an alkenyl group, as defined above, that links at least two other groups, i.e., divalent hydrocarbon groups. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Non-limiting examples of alkenylene groups include ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, s-butenylene, pentenylene, and hexenylene. Alkenylene groups can be substituted or unsubstituted.

[0045] "Alkynyl" means a straight or branched chain hydrocarbon having at least two carbon atoms and at least one triple bond. Alkynyl can contain any number of carbons (C, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C 2-20 , C 2-30 , C 2-40 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6, and C6). Non-limiting examples of alkynyl groups include acetylenyl, propynyl, 1-butynyl, 2-butynyl, butadinyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. The alkynyl group can be substituted or unsubstituted.

[0046] "Alkynylene" refers to an alkynyl group, as defined above, that links at least two other groups, i.e., divalent hydrocarbon groups. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. Non-limiting examples of alkynylene groups include ethynylene, propynylene, isopropynylene, butynylene, s-butynylene, pentynylene, and hexynylene. Alkynylene groups can be substituted or unsubstituted.

[0047] "Cycloalkyl" means a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic structure containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can contain any number of carbons (C 3-6 , C 4-6 , C 5-6 , C 3-8 , C 4-8 , C 5-8 , C 6-8 , C 3-9 , C 3-10 , C 3-11 , and C 3-12and the like). Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norborane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. The cycloalkyl group may be partially unsaturated with one or more double or triple bonds in the ring. Non-limiting examples of representative partially unsaturated cycloalkyl groups include cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-, and 1,5-isomers), norborene, and norbornadiene. When the cycloalkyl is a saturated monocyclic C 3-8 When it is cycloalkyl, non-limiting examples of representative groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. 3-6 When it is cycloalkyl, non-limiting examples of representative groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted.

[0048] "Heterocycloalkyl" refers to a saturated ring system having 3-12 ring members and 1-4 heteroatoms that are N, O, and S. The heteroatoms may be oxidized (non-limiting examples of which are -S(O)- and -S(O)2-). Heterocycloalkyl groups can contain any number of ring atoms (e.g., 3-6, 4-6, 5-6, 3-8, 4-8, 5-8, 6-8, 3-9, 3-10, 3-11, or 3-12 ring members). Heterocycloalkyl groups can contain any suitable number of heteroatoms (e.g., 1, 2, 3, or 4, or 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4). Heterocycloalkyl groups can include the following groups: aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3-, and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Heterocycloalkyl groups can also be fused to aromatic or non-aromatic ring systems to form various members, a non-limiting example of which is indoline. Heterocycloalkyl groups can be unsubstituted or substituted. For example, heterocycloalkyl groups can be C 1-6 It can be substituted with alkyl or oxo (=O), but there are many other substituents.

[0049] "Aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can contain any suitable number of ring atoms (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, as well as 6-10, 6-12, or 6-14 ring members). Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or joined by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has a methylene linking group. Some aryl groups have 6-12 ring members (such as phenyl, naphthyl, or biphenyl). Other aryl groups have 6-10 ring members (such as phenyl or naphthyl). Some other aryl groups have 6 ring members (such as phenyl). Aryl groups can be substituted or unsubstituted.

[0050] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring system containing 5-16 ring atoms, of which 1-5 ring atoms are heteroatoms (such as N, O, or S). The heteroatoms may be oxidized (non-limiting examples of which are -S(O)- and -S(O)2-). Heteroaryl groups can contain any number of ring atoms (e.g., 5-6, 3-8, 4-8, 5-8, 6-8, 3-9, 3-10, 3-11, or 3-12 ring members). Heteroaryl groups can contain any suitable number of heteroatoms (e.g., 1, 2, 3, 4, or 5, or 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5). Heteroaryl groups can have 5 to 8 ring members and 1 to 4 heteroatoms, or 5 to 8 ring members and 1 to 3 heteroatoms, or 5 to 6 ring members and 1 to 4 heteroatoms, or 5 to 6 ring members and 1 to 3 heteroatoms. Groups that can be included in heteroaryl groups include pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene, furan, thiazole, furan, thiazole, isothiazole, oxazole, and isoxazole. Heteroaryl groups can also be fused to aromatic ring systems (such as phenyl rings) to form a variety of members, non-limiting examples of which include benzopyrroles (such as indole and isoindole), benzopyridines (such as quinoline and isoquinoline), benzopyrazines (quinoxaline), benzopyrimidines (quinazoline), benzopyridazines (such as phthalazine and cinnoline), benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by bonds (such as bipyridine). Heteroaryl groups can be substituted or unsubstituted.

[0051] "Alkoxy" refers to an alkyl group with one oxygen atom connecting the alkyl group to the point of attachment, i.e., alkyl-O-. As with alkyl groups, alkoxy groups can have any suitable number of carbon atoms (C1-6 Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, s-butoxy, t-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with a variety of substituents, as described herein. Alkoxy groups can be substituted or unsubstituted.

[0052] "Hydroxyl" means an --OH functional group.

[0053] "Halogen" means fluorine, chlorine, boron and iodine.

[0054] "Fluorophore" refers to a compound that emits light, typically in the range of 300-700 nm, after excitation. A fluorophore becomes excited when it absorbs transferred light energy (e.g., a photon). When the molecule escapes from the excited state, it releases light energy in the form of lower energy photons (e.g., fluoresces), returning the dye molecule to its ground state. A fluorophore can be a natural or synthetic compound. Non-limiting examples of fluorophores include DAPI, ethidium bromide, acridine orange, GFP, mCherry, hydroxycoumarin, fluorescein, LysoTracker (red and green), Dextran-Alexa Fluor 488, Premo™ autophagy sensor LC3B-GFP, and Ac-DEVD-AMC.

[0055] "Photosensitizer" refers to a compound that upon activation by light can generate reactive groups for photodynamic therapy, typically reactive oxygen species (ROS), but can also generate reactive groups for polymerization, crosslinking, or decomposition. Photosensitizers may be useful in treating disease by generating singlet oxygen to damage tumors. Non-limiting examples of photosensitizers include porphyrins, dyes, and chlorophylls.

[0056] "Porphyrin" refers to the following porphyrin core: [ka] (wherein the porphyrin core may be substituted or unsubstituted)

[0057] "Sterols" have the following core structure: [ka] (wherein the core may be further substituted).

[0058] "Drug" refers to an agent capable of treating and / or ameliorating a condition or disease. A drug can be a hydrophobic drug (any drug that repels water). Non-limiting examples of hydrophobic drugs useful in the present invention include hydrochloroquine (HCQ), Lys05, bortezomib, β-lapachone, JQ1, napabucasin, rapamycin, paclitaxel, SN38, etoposide, lenalidomide, and apoptozole. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids (such as vinblastine and vincristine). Drugs of the present invention also include prodrug forms. One of skill in the art will recognize that other drugs are useful in the present invention.

[0059] "Imaging agent" or "contrast agent" refers to a compound that enhances the contrast of a structure in a cell or body location for imaging procedures, non-limiting examples of which include MRI, PET, SPECT, and CT. Imaging agents can emit radiation, fluorescence, magnetic fields, or radio waves. Non-limiting examples of imaging agents include radioactive metal chelators, radioactive metal atoms or ions, and fluorophores.

[0060] "Chemotherapeutic agent" refers to a chemical drug that can be used to treat a disease, non-limiting examples of which are cancer, tumors, and neoplasms. In some embodiments, the chemotherapeutic agent can be in the form of a prodrug that can be activated to a cytotoxic form. Chemotherapeutic agents are generally known to those of skill in the art and can be used in the present invention. Non-limiting examples of chemotherapeutic agents include daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoptozole, carboplatin, cisplatin, oxaliplatin, vinblastine, and vincristine.

[0061] "Molecularly targeted agents" refer to agents that can target specific molecules involved in the evolution, growth, and spread of tumors and cancers. Targeting specific molecules involved in the evolution of tumors and cancers can kill tumors and cancers or inhibit the growth and spread of tumors and cancers. Non-limiting examples of molecularly targeted agents include trastuzumab, erlotinib, imatinib, nilotinib, and vemurafenib.

[0062] "Immunotherapeutic agent" refers to a type of drug that can alter the immune response by stimulating or suppressing the immune system. Non-limiting examples of immunomodulatory agents include HCQ, Lys05, JQ1, rapamycin, napabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0063] "Radiotherapy agent" refers to an agent that can be used to treat a disease using radiation therapy. Radiation therapy is a disease treatment method that uses radiation to kill or inhibit tumor and cancer cells. Non-limiting examples of radiotherapy agents include β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, and tirapazamine.

[0064] "Nanocarrier" or "nanoparticle" refers to micelles resulting from the aggregation of the compounds of the invention. Nanocarriers of the invention can have a hydrophobic core and a hydrophilic exterior.

[0065] "Inhibit," "inhibit," and "inhibitor" refer to a compound that blocks or a method of blocking a specific action or function.

[0066] "Treate," "treating," and "treatment" refer to any indication of success in treatment or amelioration of an injury, condition, state, or symptom (e.g., pain), including improvement in any objective or subjective parameter (such as mitigation; remission; reducing the symptoms or making the symptom, injury, condition, or state more tolerable to the patient; reducing the frequency or duration of the symptom or condition; or, in some circumstances, preventing the onset of the symptom). The treatment or amelioration of a symptom can be based on any objective or subjective parameter; parameters include, for example, the results of a physical exam.

[0067] "Disease" refers to abnormal cellular function in an organism and is not the direct result of physical or external injury. Disease refers to any condition that causes anxiety, dysfunction, disability, abnormality, infection, pain, or even death. Non-limiting examples of disease include genetic diseases (such as genetic and non-genetic diseases), infectious diseases, non-infectious diseases (such as cancer, deficiency diseases, and physiological diseases).

[0068] By "administering" is meant oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, or subcutaneous administration, intrathecal administration, or implantation of a sustained release device (e.g., a mini-osmotic pump) to a subject.

[0069] "Subject" means an animal, such as a mammal, non-limiting examples of which include primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human.

[0070] "Therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refers to a dose that is administered to produce a therapeutic effect. The exact dose will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0071] "Target" or "targeting" means using a compound, protein, or antibody that specifically or selectively binds to a cell, viral particle, viral protein, antigen, or biomolecule, or localizes to a particular cell type, tissue type, microbial type, or viral type.

[0072] "Imaging" refers to the use of a device external to the subject to define the location of an imaging agent (such as the compound of the present invention). Non-limiting examples of imaging tools include positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), and X-ray computed tomography (CT). Positron emission tomography detects radiation from the emission of positrons by an imaging agent. III.Compound

[0073] In some embodiments, the present invention provides a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-4 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c W is substituted with C 3-12 Cycloalkyl, C 6-12 aryl, or 5-12 membered heteroaryl having 1-4 heteroatoms each independently being N, O, or S, wherein each cycloalkyl, aryl, and heteroaryl is optionally selected from the group consisting of C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 alkynyl; each L is independently absent, C 1-20 Alkylene, C 2-20 Alkenylene, or C 2-20 each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic agent, a sterol, C 3-12 cycloalkyl, 3-12 membered heterocycloalkyl having 1-4 heteroatoms each independently being N, O, or S; C 6-12 aryl, 5-12 membered heteroaryl having 1-4 heteroatoms each independently being N, O, or S, -OH, or -NH; R 1a is C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-40Alkoxy, hydroxyl, or -NR 1b R 1c is substituted with;R 1b is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 Alkynyl; R 1c is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 alkynyl, or -LW; R 2a , R 2b , R 3a , and R 3b are each independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 alkoxy, halogen, -CN, or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; each X is independently absent or -O - However, X is absent and R 2a and R 2b is hydrogen, R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, boron, or -NO2, R 1 is C 2-40 Alkyl, C 2-40 Alkenyl, C 4-40 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a , X is absent, and R 1 is -CH2CH2NH(7-chloro-4-quinolinyl), R 2a and R 2b When is hydrogen, R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 It is selected from alkoxy, fluorine, boron, iodine, -CN, or -NO2.

[0074] In some embodiments, the present invention provides a compound of formula (I), 1 is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c W is substituted with C 3-12 Cycloalkyl, C 6-12 Aryl, or C 4-12 and heteroaryl, wherein each cycloalkyl, aryl, and heteroaryl is optionally selected from the group consisting of C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl; each L is independently absent, C 1-10 Alkylene, C 2-10 Alkenylene, or C 2-10 each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic agent, or a sterol; R 1a is C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c is replaced by R 1b is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl, R 1c is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C2-20 alkynyl, or -LW; R 2a , R 2b , R 3a , and R 3b are each independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, halogen, -CN, or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; each X is independently absent or -O - However, X is absent and R 2a and R 2b is hydrogen, R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, boron, or -NO2, R 1 is C 2-20 Alkyl, C 2-20 Alkenyl, C 4-20 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a ; X is absent and R 1 is -CH2CH2NH(7-chloro-4-quinolinyl), R 2a and R 2b When is hydrogen, R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, fluorine, boron, iodine, -CN, or -NO2).

[0075] In some embodiments, R 1 is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20Alkoxy, hydroxyl, or -NR 1b R 1c W is substituted with C 3-12 Cycloalkyl, C 6-12 aryl, or 5-12 membered heteroaryl, which 5-12 membered heteroaryl has 1-4 heteroatoms of N, O, and S, and each cycloalkyl, aryl, and heteroaryl is optionally C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl; each L is independently absent, C 1-10 Alkylene, C 2-10 Alkenylene, or C 2-10 alkynylene; each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic agent, or a sterol; R 1a is C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c is substituted with;R 1b is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 Alkynyl; R 1c is hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 alkynyl, or -LW; R 2a , R 2b , R 3a , and R 3b are each independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20alkoxy, halogen, -CN, or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; each X is independently absent or -O - However, X is absent and R 2a and R 2b is hydrogen, R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, boron, or -NO2, when R 1 is C 2-20 Alkyl, C 2-20 Alkenyl, C 4-20 Alkynyl, -W, -(LY) p -Z, or C(O)R 1a , X is absent, and R 1 is -CH2CH2NH(7-chloro-4-quinolinyl), R 2a and R 2b When is hydrogen, R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 It is selected from alkoxy, fluorine, boron, iodine, -CN, or -NO2.

[0076] In some embodiments, R 1 is hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, -W, -(LY) p -Z, or -C(O)R 1a In some embodiments, R 1 is C 1-40 Alkyl, C 2-40 Alkenyl, -(LY) p -Z, or -C(O)R 1a It is.

[0077] In some embodiments, R 1 is C 1-40 In some embodiments, R 1 is C 1-25In some embodiments, R 1 is C 1-20 In some embodiments, R 1 is C 10-25 In some embodiments, R 1 is C 10-20 In some embodiments, R 1 is C 12-22 In some embodiments, R 1 is C 12-18 It is an alkyl.

[0078] In some embodiments, R 1 is C 2-40 In some embodiments, R is alkenyl. 1 is C 20-40 In some embodiments, R is alkenyl. 1 is C 30-40 In some embodiments, R is alkenyl. 1 is C 2-40 In some embodiments, R 1 is C 20-40 In some embodiments, R 1 is C 30-40 It is alkynyl.

[0079] In some embodiments, R 1 is W. In some embodiments, W is C 3-12 Cycloalkyl, C 6-12 aryl, or 5-12 membered heteroaryl having 1-4 heteroatoms each independently being N, O, or S, wherein each cycloalkyl, aryl, and heteroaryl is optionally selected from the group consisting of C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 In some embodiments, W is substituted with C 5-12 Cycloalkyl, C 6-12aryl, or 5-12 membered heteroaryl having 1-4 heteroatoms, each independently being N, O, or S. In some embodiments, W is C 5-12 In some embodiments, W is C 5-8 In some embodiments, W is cyclopentyl or cyclohexyl.

[0080] In some embodiments, R 1 HA-(LY) p In some embodiments, p is an integer from 1 to 20. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is 1, 2, 3, 4, or 5. In some embodiments, p is 1.

[0081] In some embodiments, L is C 1-20 Alkylene, C 2-20 Alkenylene, or C 2-20 In some embodiments, L is C 1-20 In some embodiments, L is C 1-10 In some embodiments, L is C 1-5 It is alkylene.

[0082] In some embodiments, Y is absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-. In some embodiments, Y is absent, -NH-, -NHC(O)-, -NHC(O)NH-, -OC(O)-, -OC(O)NH-, or -C(O)-. In some embodiments, Y is absent, -NH-, -NHC(O)-, or -NHC(O)NH-. In some embodiments, Y is absent, -NH-, or -NHC(O)NH-.

[0083] In some embodiments, Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic agent, a sterol, C3-12 cycloalkyl, 3-12 membered heterocycloalkyl having 1-4 heteroatoms, each independently being N, O or S; C 6-12 aryl, a 5-12 membered heteroaryl having 1-4 heteroatoms each independently being N, O, or S, -OH, or -NH2.

[0084] Non-limiting examples of photosensitizers useful in the present invention include porphyrins, benzoporphyrins, corrins, chlorins, bacteriochlorophylls, corphins, or derivatives thereof. Representative photosensitizers are shown below: [ka] [ka]

[0085] In some embodiments, the photosensitizer is a porphyrin, a benzoporphyrin, a corrin, a chlorin, a bacteriochlorophyll, a korufin, or a derivative thereof. In some embodiments, the photosensitizer compound is a porphyrin, a pyropheophorbide-a, a pheophorbide, a chlorin e6, a purpurin, a purpurinimide, a verteporfin, a photofrin porfimer, a rostaporfin, a talporfin, or a temoporfin. In some embodiments, the photosensitizer is a pyropheophorbide-a. In some embodiments, the photosensitizer is a pheophorbide-a. In some embodiments, the photosensitizer is a porphyrin.

[0086] Any suitable porphyrin can be used in the compounds of the present invention.Non-limiting examples of representative porphyrins suitable for the present invention include pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, or purpurinimide.In some embodiments, the porphyrin can be pheophorbide-a.In some embodiments, the porphyrin can be pyropheophorbide-a.

[0087] In some embodiments, Z is a porphyrin, a sterol, a 6-12 membered heterocycloalkyl, an 8-12 membered heteroaryl, -OH, or -NH2, wherein the 6-12 membered heterocycloalkyl and the 8-12 membered heteroaryl have 1-4 heteroatoms of N, O, and S. In some embodiments, Z is a porphyrin, cholic acid, indoline, isoindoline, 1-isoindolinone, pthalimid, phthalic anhydride, -OH, or -NH2.

[0088] In some embodiments, p is 1; L is C 4-5 is alkylene; Y is absent, -NH-, or -NHC(O)-; and Z is porphyrin, cholic acid, isoindoline, phthalimide, -OH, or -NH2.

[0089] In some embodiments, R 1 HA-C(O)R 1a In some embodiments, R 1a is C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-40 Alkoxy, hydroxyl, or -NR 1b R 1c In some embodiments, R 1a is C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 alkynyl, wherein each alkyl, alkenyl, and alkynyl is optionally selected from the group consisting of C 1-20 Alkoxy, hydroxyl, or -NR 1b R 1c In some embodiments, R 1a is C 1-10 Alkyl, C 2-10 Alkenyl, or C 2-10 In some embodiments, R 1a is C 1-10In some embodiments, R 1a is C 1-5 In some embodiments, R 1a is methyl, ethylene, propyl, or butyl.

[0090] In some embodiments, R 1b is C 1-40 Alkyl, C 2-40 Alkenyl, or C 2-40 In some embodiments, R 1b is C 1-20 Alkyl, C 2-20 Alkenyl, or C 2-20 In some embodiments, R 1b is C 1-10 In some embodiments, R 1b is C 1-5 In some embodiments, R 1b is methyl, ethylene, propyl, or butyl.

[0091] In some embodiments, R 1c is C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 alkynyl, or -LW. In some embodiments, R 1c is C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 alkynyl, or -LW. In some embodiments, R 1c is C 1-10 In some embodiments, R 1c is C 1-5 In some embodiments, R 1c is methyl, ethylene, propyl, or butyl.

[0092] In some embodiments, R 2a and R 2b are each independently hydrogen, C 1-20 Alkyl, C 2-20Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, halogen, -CN, or -NO2. In some embodiments, R 2a and R 2b are each independently hydrogen, C 1-20 In some embodiments, R 2a and R 2b are each independently hydrogen or halogen. In some embodiments, R 2a and R 2b are each independently hydrogen, fluorine, chlorine, boron, or iodine. In some embodiments, R 2a and R 2b are each independently hydrogen.

[0093] In some embodiments, R 3a and R 3b are each independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, halogen, -CN, or -NO2. In some embodiments, R 3a and R 3b are each independently hydrogen, C 1-20 In some embodiments, R 3a and R 3b are each independently hydrogen or halogen. In some embodiments, R 3a and R 3b are each independently hydrogen, fluorine, chlorine, boron, or iodine. In some embodiments, R 3a and R 3b are each independently hydrogen.

[0094] In some embodiments, m and n are independently integers from 1 to 10. In some embodiments, m and n are independently integers from 1 to 5. In some embodiments, m and n are independently 1, 2, 3, 4, or 5. In some embodiments, m and n are each independently 1.

[0095] In some embodiments, each X is independently absent or -O - In some embodiments, each X is absent. In some embodiments, each X is -O - is - .

[0096] In some embodiments, each X is absent. In some embodiments, the compound has formula (Ia): [ka] It is.

[0097] In some embodiments, R 1 is C 1-20 alkyl, and the compound has the formula (Ia): [ka] It is.

[0098] In some embodiments, the compound has the structure: [ka] (where n is an integer from 1 to 7).

[0099] In some embodiments, the compound is [ka] is selected from the group consisting of:

[0100] In some embodiments, the compound is [ka] It is.

[0101] In some embodiments, the compound is [ka] is selected from the group consisting of:

[0102] In some embodiments, each X is -O - In some embodiments, the compound is a compound of formula (Ib): [ka] It is.

[0103] In some embodiments, the compound is [ka] is selected from the group consisting of:

[0104] In some embodiments, the compound is [ka] [ka] is selected from the group consisting of:

[0105] The present invention includes all tautomers and stereoisomers of the compounds of the present invention, either in the form of a mixture or in pure or substantially pure form. The compounds of the present invention may have asymmetric centers at the carbon atoms, and therefore the compounds of the present invention may exist in diastereomeric or enantiomeric forms, or in mixtures thereof. All conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic mixtures, diastereomeric mixtures, and other mixtures of such isomers, as well as solvates, hydrates, isomorphs, polymorphs, and tautomers, are included within the scope of the present invention. The compounds according to the present invention may be prepared using diastereomers, enantiomers, or racemic mixtures as starting materials. Furthermore, diastereomeric and enantiomeric products may be separated by chromatography, fractional crystallization, or other methods known to those skilled in the art.

[0106] The present invention also includes isotopically labeled compounds of the present invention, in which one or more atoms have been replaced with one or more atoms having a particular atomic mass or mass number. Non-limiting examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, sulfur, and chlorine (e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 18 F, 35 S, and 36 Isotopically labeled compounds of the invention are useful in tissue distribution assays of the compounds and their prodrugs and metabolites; preferred isotopes for such assays include 3 H and 14 C. In addition, in some cases, heavier isotopes (deuterium ( 2 H)) can provide greater metabolic stability, which confers therapeutic benefits such as increased in vivo half-life or reduced dose requirements. Isotopically labeled compounds of the invention can generally be prepared by replacing a non-isotopically labeled reagent with an isotopically labeled reagent according to methods known to those of skill in the art. Compounds of the invention can be isotopically labeled at basic amines in the aromatic ring and at positions adjacent to the methyl group of the methoxy substituent.

[0107] The compounds of the present invention can also be in the form of pharma- ceutically acceptable salts, such as acid salts or base salts of the compounds of the present invention. Representative examples of pharma-ceutically acceptable salts are salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (such as fumaric acid, acetic acid, propionic acid, glutamic acid, citric acid, etc.), and salts of quaternary ammonium (such as methyl iodide, ethylene iodide, etc.). It is understood that pharma-ceutically acceptable salts are non-toxic. Additional information regarding suitable pharma-ceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985 (incorporated herein by reference). IV. Nanocarriers

[0108] In some embodiments, the invention provides nanocarriers having an interior and an exterior, which comprise a plurality of compounds of the invention, or pharma- ceutically acceptable salts thereof, each of which, when self-assembled in an aqueous medium, forms a nanocarrier such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0109] The diameter of nanocarriers of the present invention can be any suitable size. In some embodiments, nanocarriers can have a diameter of 5-200 nm. In some embodiments, nanocarriers can have a diameter of 10-150 nm. In some embodiments, nanocarriers can have a diameter of 50-150 nm. In some embodiments, nanocarriers can have a diameter of 100-150 nm. In some embodiments, nanocarriers can have a diameter of about 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, or 130 nm. In some embodiments, nanocarriers can have a diameter of about 100 nm.

[0110] The exterior of the nanocarrier can be utilized to target cells or lysosomes. The nanocarrier can target cells or lysosomes to inhibit autophagy. In some embodiments, the nanocarrier can target lysosomal disruption, lysosomal dysfunction, autophagy inhibition, or a combination thereof. In some embodiments, the nanocarrier targets lysosomes.

[0111] In some embodiments, the hydrophobic pocket is R 1 In some embodiments, the nanocarrier comprises one or more hydrophobic drugs or imaging agents sequestered in a hydrophobic pocket of the nanocarrier.

[0112] The hydrophobic drug useful in the present invention can be any hydrophobic drug known to those skilled in the art.Non-limiting examples of hydrophobic drugs useful in the present invention include deoxycholic acid, deoxycholate, resiquimod, gardiquimod, imiquimod, taxanes (e.g. paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, Hongdoushan A, Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epoterone class), rapamycin, and platinum drugs.Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids (such as vinblastine and vincristine).

[0113] Non-limiting examples of other hydrophobic drugs useful in the present invention include chemotherapeutic agents, molecular targeted agents, immunomodulatory agents, immunotherapeutic agents, radiotherapeutic agents, or combinations thereof.

[0114] In some embodiments, the hydrophobic drug is a chemotherapeutic agent, a molecular targeting agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof. In some embodiments, the hydrophobic drug is an immunotherapeutic agent. Non-limiting examples of immunotherapeutic agents useful in the present invention include HCQ, Lys05, JQ1, rapamycin, napabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0115] In some embodiments, the hydrophobic drug is a radiotherapeutic agent. Non-limiting examples of radiotherapeutic agents useful in the present invention include β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, and tirapazamine.

[0116] In some embodiments, the hydrophobic drug is a chemotherapeutic agent or a molecular targeted agent. Non-limiting examples of chemotherapeutic agents or molecular targeted agents include daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoptozole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, and vemurafenib.

[0117] In some embodiments, the hydrophobic drug is an FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, an inhibitor of checkpoint-1 or 2, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, butabulin, ofatumumab, zanolibumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, Cilengitide, Dimatecan, IL13-PE38QQR;INO 1001, IPdR1 KRX-0402, Lucanton, LY 317615, Neurajiab, Vitespan, Rta 744, Sdx 102, Talampanel, Atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzo ... yl]-L-glutamic acid disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogens, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperazinemethyl)-indolyl-quinolone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t) 6, Azgly 10] acetate (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84N 18 O 14-(C2H4O2)X (where x=1-2.4)], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, ionafarnib, BMS-214662, tipifarnib ;Amifostine, NVP-LAQ824, suberoylanilide hydroxamate, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilben Bestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab , streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovastat, BMS-275291,Squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin difitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine , bevacizumab (monoclonal antibody) and Erbitux, Cremophor-free paclitaxel, Epithilon B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte-colony-stimulating factor, zoledronate, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon alpha-2a, interferon alpha-2a, pegylated interferon alpha-2b, interferon alpha-2b, interferon alpha-2c, interferon alpha-2d, interferon alpha-2e, interferon alpha-2f, interferon alpha-2g ... Terferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibritugomab tiuxetan, androgen, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium-89, casopitant, netupitant,NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspeg filgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib, or combinations thereof. In some embodiments, the hydrophobic drug is HCQ, Lys05, JQ1, rapamycin, napabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, tirapazamine, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoptozole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, vemurafenib, or a combination thereof.

[0118] In some embodiments, the nanocarriers comprise a plurality of compounds of the invention having the structures of the compounds described above. V. Formulation and Administration

[0119] The compounds, nanocarriers, and compositions of the present invention can be prepared in a variety of oral, parenteral, and topical dosage forms. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by the patient. The compositions of the present invention can also be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally. The compositions described herein can also be administered by inhalation (e.g., intranasally). In addition, the compositions of the present invention can be administered transdermally. The compositions of the invention may also be administered by ocular, vaginal, and rectal routes, examples of which include suppositories, insufflation, powders, and aerosol formulations (e.g., steroid inhalants: see Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995). Thus, the invention also provides pharmaceutical compositions comprising a pharma- ceutically acceptable carrier or excipient and a compound of the invention.

[0120] To prepare pharmaceutical compositions from the compounds of the present invention, the pharma- ceutical acceptable carrier can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier can be one or more substances, which can also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials. Details regarding formulation and administration techniques are well described in the academic and patent literature. See, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton, Pa. ("Remington's").

[0121] In powders, the base is a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a base having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain from 5% or 10% to 70% of the compound of the invention.

[0122] Non-limiting examples of suitable solid excipients include magnesium carbonate, magnesium stearate, talc, pectin, dextrin, starch, tragacanth gum, low melting wax, cocoa butter, carbohydrates, sugars (non-limiting examples of which include lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants), celluloses (such as methylcellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose), and gums (including gum arabic and gum tragacanth), as well as proteins (non-limiting examples of which include gelatin and collagen). If desired, disintegrants or solubilizers can be added (such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof (such as sodium alginate)).

[0123] The dragee cores are provided with a suitable coating, such as concentrated sugar solutions (which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide), lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings to identify the product or to characterize the amount of active compound (i.e., the dose). The pharmaceutical preparations of the invention can also be used orally, for example in push-fit capsules made of gelatin, as well as in soft, sealed capsules made of gelatin and a coating (such as glycerol or sorbitol). Push-fit capsules can contain the compound of the invention mixed with fillers or binders (such as lactose or starch), lubricants (such as talc or magnesium stearate), and optionally stabilizers. In soft capsules, the compound of the invention can be dissolved or suspended in a suitable liquid (fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0124] For preparing suppositories, a low melting wax (such as a mixture of fatty acid glycerides or cocoa butter) is first melted and the compound of the present invention is dispersed homogeneously therein, for example by stirring, The molten homogeneous mixture is then poured into suitable sized molds and allowed to cool and solidify.

[0125] Liquid form preparations include solutions, suspensions, and emulsions (for example, water or water / propylene glycol solutions).For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0126] Aqueous solutions suitable for oral use can be prepared by dissolving the compounds of the present invention in water and adding suitable colorants, flavorings, stabilizers and thickeners, if desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active ingredient in water with a viscous material (such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic) and a dispersing or wetting agent (natural phosphatides (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide and partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitol monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The osmolarity of the preparation may be adjusted.

[0127] It includes solid form preparations that are intended to be converted shortly before use into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial or natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0128] Oil suspensions can be formulated by suspending the compounds of the present invention in a vegetable oil (such as peanut oil, olive oil, sesame oil, or coconut oil), or a mineral oil (such as liquid paraffin); or a mixture thereof. Oil suspensions can contain a thickening agent (such as beeswax, hard paraffin, or cetyl alcohol). Sweeteners can be added to provide a palatable oral preparation (such as glycerol, sorbitol, or sucrose). These preparations can be preserved by the addition of an antioxidant (such as ascorbic acid). For an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical preparations of the present invention can also be in the form of oil-in-water emulsions. The oil phase can be a vegetable oil or a mineral oil as described above, or a mixture thereof. Suitable emulsifying agents include natural gums (such as gum arabic and gum tragacanth), natural phosphatides (such as soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate), and condensation products of these partial esters with ethylene oxide (such as polyoxyethylene sorbitan monooleate). Emulsions can also contain sweetening and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, preservative, or coloring agent.

[0129] In another embodiment, the compositions of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a cavity or lumen of an organ. The formulation for administration will generally comprise a solution of the compositions of the present invention in a pharma- ceutically acceptable base. Acceptable vehicles and solvents that can be used include water and Ringer's solution, isotonic sodium chloride. In addition, sterile, fixed oils can usually be used as a solvent or suspending medium. For this purpose, any bland, fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, can be used to prepare injectable formulations as well. These solutions are sterile and generally free of undesirable substances. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations can contain pharma-ceutically acceptable auxiliary substances necessary to mimic physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate. The concentration of the composition of the present invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the specific mode of administration selected and the needs of the patient. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. The suspension can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.

[0130] The compositions of the present invention can be delivered by any suitable means, including oral, parenteral, and topical. Topical transdermal administration can be formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0131] The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is divided into unit doses containing appropriate amounts of the compound of the present invention. The unit dosage form can be a packaged preparation, a package containing discrete amounts of the preparation (such as packaged tablets, capsules, and powders in vials or ampoules). The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or the appropriate number of any of these packaged forms.

[0132] The compounds of the invention and nanocarriers can be present in any suitable amount, which may depend on a variety of factors, including, but not limited to, the weight and age of the subject, the disease state, etc. Suitable dose ranges of the compounds of the invention include about 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable doses of the compounds of the invention include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg.

[0133] The compounds of the invention and nanocarriers can be administered at any suitable frequency, interval, and duration. For example, the compounds of the invention can be administered once per hour, or twice, three or more times per hour, once per day, or twice, three or more times per day, or once every 2, 3, 4, 5, 6, or 7 days to provide a preferred dosage level. When the compounds of the invention are administered more than once per day, representative intervals include 5, 10, 15, 20, 30, 45, and 60 minutes, as well as 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24 hours. The compounds of the invention can be administered once, twice, or three times, or more times, for 1 hour, 1-6 hours, 1-12 hours, 1-24 hours, 6-12 hours, 12-24 hours, only 1 day, 1-7 days, only 1 week, 1-4 weeks, 1 month, 1-12 months, over a year, or even indefinitely.

[0134] The compositions may also contain other compatible therapeutic agents. The compounds described herein may be used in combination with each other, with other active agents known to be useful for modulating the glucocorticoid receptor, or with auxiliary agents that may not be effective alone but may contribute to the effect of the active agent.

[0135] The compound of the present invention can be administered simultaneously with another active agent. Simultaneous administration includes administration of the compound of the present invention and the active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of each other. Simultaneous administration also includes administration of the compound of the present invention and the active agent at the same time, at about the same time (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Furthermore, the compound of the present invention and the active agent can each be administered once a day, or two, three, or more times a day to provide a preferred daily dosage level.

[0136] In some embodiments, simultaneous administration can be achieved by co-formulation, i.e., preparing a single pharmaceutical composition that contains both the compound of the invention and the active agent, hi other embodiments, the compound of the invention and the active agent can be formulated separately.

[0137] The compounds of the invention and the active agent can be present in the compositions of the invention in any suitable weight ratio, such as about 1:100 to about 100:1 (w / w), or about 1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1:5 to about 5:1 (w / w). The compounds of the invention and the other active agent can be present in any suitable weight ratio, such as about 1:100 (w / w), 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, or 100:1 (w / w). Other doses and dose ratios of the compounds of the invention and the active agents are suitable for the compositions and methods of the invention. VI. Treatment Methods

[0138] In some embodiments, the present invention provides a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the present invention.

[0139] In some embodiments, the method further comprises combination therapy with an additional agent to treat the disease. The additional agent is a therapeutic agent. A non-limiting example of a combination therapy of the invention includes the use of the nanocarriers of the invention and one or more additional agents.

[0140] Non-limiting examples of combination therapies include immunotherapy, radiation therapy, chemotherapy, targeted therapy, or a combination thereof.

[0141] In some embodiments, the method further comprises one or more additional agents, which are chemotherapeutic agents, molecular targeted agents, immunotherapeutic agents, radiotherapeutic agents, or combinations thereof. In some embodiments, the additional agent is an immunotherapeutic agent. Immunotherapeutic agents useful in the present invention are set forth above. In some embodiments, the additional agent is a radiotherapeutic agent. Radiotherapeutic agents useful in the present invention are set forth above. In some embodiments, the additional agent is a chemotherapeutic agent or a molecular targeted agent. Chemotherapeutic agents and molecular targeted agents useful in the present invention are set forth above.

[0142] In some embodiments, the one or more additional agents include two additional agents. In some embodiments, the additional agents are an immunotherapeutic agent and a radiotherapeutic agent. In some embodiments, the additional agents are an immunotherapeutic agent and a chemotherapeutic agent. In some embodiments, the additional agents are an immunotherapeutic agent and a molecular targeted agent. In some embodiments, the additional agents are a radiotherapeutic agent and a chemotherapeutic agent. In some embodiments, the additional agents are a radiotherapeutic agent and a molecular targeted agent.

[0143] In some embodiments, the additional agent is an FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, an inhibitor of checkpoint-1 or 2, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, butabulin, ofatumumab, zanolibumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, Cilengitide, Dimatecan, IL13-PE38QQR;INO 1001, IPdR1 KRX-0402, Lucanton, LY 317615, Neurajiab, Vitespan, Rta 744, Sdx 102, Talampanel, Atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl l]-L-glutamic acid disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogens, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperazinemethyl)-indolyl j-quinolone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t) 6, Azgly 10] acetate (Pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H84 N 18 O 14-(C2H4O2)X (where x=1-2.4)], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, ionafarnib, BMS-214662, tipifarnib ;Amifostine, NVP-LAQ824, suberoylanilide hydroxamate, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilben Bestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab , streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovastat, BMS-275291,Squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin difitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine , bevacizumab (monoclonal antibody) and Erbitux, Cremophor-free paclitaxel, Epithilon B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte-colony-stimulating factor, zoledronate, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon alpha-2a, interferon alpha-2a, pegylated interferon alpha-2b, interferon alpha-2b, interferon alpha-2c, interferon alpha-2d, interferon alpha-2e, interferon alpha-2f, interferon alpha-2g ... Terferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibritugomab tiuxetan, androgen, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium-89, casopitant, netupitant,NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspeg filgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib, or combinations thereof. In some embodiments, the additional agent is HCQ, Lys05, JQ1, rapamycin, napabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, tirapazamine, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoptozole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, vemurafenib, or a combination thereof.

[0144] Diseases that may be treated by the methods of the invention include coronavirus, malaria, antiphospholipid syndrome, lupus, rheumatoid arthritis, chronic urticaria or Sjogren's disease, and cancer (non-limiting examples of which include carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma), head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma (for additional cancers, see CANCER: PRINCIPLES AND PRACTICE (DeVita, VT et al. eds 2008)).

[0145] Other diseases that can be treated by the nanocarriers of the present invention include (1) inflammatory or allergic diseases (such as systemic anaphylactic or hypersensitivity responses, drug allergies, insect bite allergies, etc.); inflammatory bowel diseases (such as Crohn's disease, ulcerative colitis, ileitis, and enteritis); vaginitis; psoriasis and inflammatory skin diseases (such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, etc.); vasculitis; spondyloarthritis; scleroderma; respiratory allergic diseases (such as asthma, allergic rhinitis, hypersensitivity pulmonary disease, etc.); and (2) autoimmune diseases (such as arthritis ( (3) graft rejection (including allograft rejection and graft-versus-host disease); and (4) other diseases in which unwanted inflammatory responses should be inhibited (e.g., atherosclerosis, myositis, neurological conditions (e.g., stroke and closed head injury), neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis media, chronic obstructive pulmonary disease, sinusitis, and Behcet's syndrome).

[0146] In some embodiments, the disease is cancer. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and uterine cancer. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, and uterine cancer.

[0147] In some embodiments, the disease is coronavirus, malaria, antiphospholipid syndrome, lupus, rheumatoid arthritis, chronic urticaria, or Sjogren's disease.

[0148] In some embodiments, the method of treating disease comprises targeting cellular autophagy and / or lysosome.Targeting autophagy can lead to autophagy inhibition or autophagy activation.Targeting lysosome can lead to lysosomal destruction, lysosomal dysfunction, or both.

[0149] In some embodiments, the therapy targets lysosomal disruption, lysosomal dysfunction, and / or autophagy inhibition, hi some embodiments, the therapy targets the lysosome.

[0150] In some embodiments, the nanocarriers target lysosomal disruption, lysosomal dysfunction, and / or autophagy inhibition, hi some embodiments, the nanocarriers target the lysosome. VII. Imaging Methods

[0151] In some embodiments, the invention provides a method of imaging comprising administering to a subject from which an image is to be obtained an effective amount of a nanocarrier of the invention.

[0152] The imaging agent useful in the present invention can be any imaging agent known to those skilled in the art. Non-limiting examples of imaging agents include paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are imaging agents that are magnetic under an externally applied field. Non-limiting examples of paramagnetic agents include iron particles, including nanoparticles. Optical probes are fluorescent compounds that can be detected by stimulating with radiation of one wavelength and detecting with radiation of a different second wavelength. Non-limiting examples of optical probes useful in the present invention include Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo radioactive decay. Non-limiting examples of radionuclides useful in the present invention include: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu, and 241 Am.

[0153] Non-limiting examples of imaging modalities useful in the present invention include fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), X-ray computed tomography (CT), echocardiography, and functional near-infrared spectroscopy. VIII. Examples Example 1: Compounds

[0154] Materials and Equipment. Chemicals (e.g., diethylenetriamine, dodecylaldehyde, fatty alcohols, pyridinium dichromate, sodium cyanoborohydride, ammonium hydroxide solution, deuterated solvents, anhydrous solvents, and Z-Arg-Arg-AMC) were purchased from Millipore-Sigma (MO, USA). 4,7-Dichloroquinoline, anhydrous sulfate, and bulk solvents were purchased from Fisher Scientific (MA, USA). All solvents were used directly without further purification. Water used in all experiments was purified with a Mill-Q filtration system. Other reagents or drugs were purchased as indicated: tridecanal (Alfa Aesar), HCQ (Specturm), Lys05 (MedchemExpress), bortezomib (eNovation chemical), DiD perchlorate and β-lapachone (Tocris Bioscience), JQ1 and napabucasin (ApExBIO), rapamycin, paclitaxel, and vinblastine (LC Laboratory), CN38 (Acros Organics), etoposide (AdipoGen), lenalidomide (Matrix Scientific), napabucasin (ApExBIO), and apoptozole (Selleck). Lysosomal enrichment kit, LysoTracker (Red & Green), acridine orange, Dextran-Alexa Fluor 488, Premo™ autophagy sensor LC3B-GFP were purchased from Thermo Fisher (MA, USA). SensoLyte® Homogeneous AMC Caspase-3 / 7 Assay Kit and FITC-Annexin V / PI Apoptosis Kit were purchased from AnaSpec (CA, USA) and Biolegend (CA, USA), respectively. Compounds were characterized by a 600 MHz NMR spectrometer (Bruker, Germany) for NMR spectra and an LTQ-Orbitrap XL Hybrid Ion Trap Mass Spectrometer (Thermo Fisher, MA, USA) for ESI-HRMS spectra.Cell imaging studies were performed with a fluorescence microscope (Olympus, Tokyo, Japan) or an LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Absorbance and fluorescence intensity were measured with a SpectraMax M2 microplate reader (Molecular Devices, CA, USA). Western blots were developed with a Power Pac 200 electrophoresis apparatus (Bio-Rad, CA, USA). Studies including WB imaging, in vivo and in vitro fluorescence imaging were performed with a ChemiDocTM MP imaging system (Bio-Rad, CA, USA). DLS experiments were performed with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). TEM was performed with a Talos L120C TEM (FEI, OR, USA) at an accelerating voltage of 80 kV. Apoptosis assays were performed with a BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Isolation of cancer stem cells was performed by BD FACSAria II cell sorter (BD Biosciences, NJ, USA). Matrigel for 3D culture (Cat# 354230) and xenograft model establishment (Cat# 354234) were both purchased from Corning (NY, USA). LC3B antibody (1:1000, catalog #2775), SQSTM1 / p62 antibody (1:1000, catalog #39749), and β-actin antibody (1:1000, catalog #4970) were purchased from Cell Signaling, Pacific Blue anti-CD44 antibody (5 μL per million cells in 100 μL volume, catalog #338823); APC anti-CD326 (EpCAM) antibody (5 μL per million cells in 100 μL volume, catalog #324207); PE / Cy7 anti-CD24 antibody (5 μL per million cells in 100 μL volume, catalog #311119) were obtained from Biolegend. [ka]

[0155] Synthesis of O-methyl-serine-dodecylamide hydrochloride (MSDH). MSDH was synthesized according to published literature (Bioorg. Med. Chem. Lett. 1995, 5, 893-898). 1 H NMR (600 MHz, CDCl3): δ 7.40 (s, 1 H), 3.63 (m, 1 H), 3.58 (m, 2 H), 3.70 (s, 3 H), 3.26 (m, 2 H), 1.74 (s, 2 H), 1.51 (m, 2 H), 1.29 (m, 18 H), 0.89 (t, 3 H, J=7.2 Hz). ESI-HRMS:C 16 H 35 N2O2 + m / z [M+H] + Calculated value: 287.2693, actual value: 287.2690. [ka]

[0156] Synthesis of BAQ. In a 10 mL flask, 4,7-dichloroquinoline (1.2 g, 6.00 mmol) was kept at 80°C without stirring for 2 hours, and then diethylenetriamine (0.22 mL, 2.00 mmol) was added. The reaction solution was stirred at 130°C for 6 hours. The residue was taken up with 30 mL of methanol to give a white solid as BAQ compound. Yield: 470 mg, 55%. 1 H NMR (600 MHz, DMSO-d6): δ 8.38 (d, 2 H, J=6.0 Hz), 8.23 ​​(d, 2 H, J=10.8 Hz), 7.78 (d, 2 H, J=1.8 Hz), 7.42 (dd, 2 H, J1=10.8 Hz, J2=2.4 Hz), 7.25 (s, 1 H), 6.51 (d, 2 H, J=6.6 Hz), 3.40 (t, 4 H, J=7.2 Hz), 2.94 (t, 4 H, J=7.8 Hz). ESI-HRMS:C22 H 22 Cl2N5 + m / z [M+H] + The calculated value was 426.1247, and the measured value was 426.1243.

[0157] General synthetic method for BAQ12-18. To a solution of BAQ (426 mg, 1.0 mmol) in 30 mL of anhydrous methanol and 10 mL of anhydrous dichloromethane, the corresponding aldehyde (2 mmol) and acetic acid (20 μL) were added, followed by stirring at room temperature for 20 min, followed by the addition of sodium cyanoborohydride (126 mg, 2 mmol). The mixture was stirred for 12 h and diluted with chloroform (100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate overnight. The crude product was purified by silica gel chromatography using an eluent containing 0.1% triethylamine (dichloromethane:methanol = 30:1 to 10:1) to give the corresponding compound. [ka]

[0158] BAQ12. Yield: 320 mg, 53.8%. 1 H NMR (600 MHz, CD3OD): δ 8.27 (d, 2 H, J=5.4 Hz), 7.67 (d, 2 H, J=2.4 Hz), 7.55 (d, 2 H, J=9.0 Hz), 6.95 (dd, 2 H, J1=9.0 Hz, J2=1.8 Hz), 6.46 (d, 2 H, J=5.4 Hz), 3.41 (t, 4 H, J=6.0 Hz), 2.90 (t, 4 H, J=6.0 Hz), 2.66 (t, 2 H, J=6.6 Hz), 1.55 (m, 2 H), 1.33 (m, 20 H), 0.91 (t, 3 H, J=7.2 Hz). 13C NMR (150 MHz, CD3OD): δ 150.9, 150.8, 147.9, 134.8, 126.3, 124.4, 121.9, 117.0, 98.4, 54.1, 5 1.9, 40.3, 31.6, 29.5, 29.5, 29.4, 29.4, 29.1, 27.4, 27.3, 22.4, 13.1. ESI-HRMS: C 34 H 46 Cl2N5 + m / z [M+H] + Calculated value: 594.3125, actual value: 594.3134.

[0159] BAQ13. Yield: 350 mg, 57.5%. 1 H NMR (600 MHz, CD3OD): δ 8.27 (d, 2 H, J=5.4 Hz), 7.67 (d, 2 H, J=1.8 Hz), 7.56 (d, 2 H, J=9.0 Hz), 6.96 (dd, 2 H, J1=9.0 Hz, J2=2.4 Hz), 6.46 (d, 2 H, J=5.4 Hz), 3.42 (t, 4 H, J=6.0 Hz), 2.90 (t, 4 H, J=6.0 Hz), 2.66 (t, 2 H, J=7.2 Hz), 1.56 (m, 2 H), 1.32 (m, 23 H), 0.92 (t, 3 H, J=7.2 Hz). 13 CNMR (150 MHz, CD3OD): δ 151.0, 150.7, 147.8, 134.8, 126.1, 124.5, 121.9, 117.0, 98.4, 54.6, 5 1.9, 40.3, 31.7, 29.5, 29.5, 29.5, 29.4, 29.1, 27.4, 27.3, 22.3, 13.1. ESI-HRMS:C 35 H 48 Cl2N5 + m / z [M+H] + Calculated value: 608.3281, measured value: 608.3274.

[0160] BAQ14. Yield: 295 mg, 47.4%. 11H NMR (600 MHz, CD3OD): δ 8.56 (d, 2 H, J = 9.0 Hz), 8.48 (d, 2 H, J = 4.8 Hz), 7.86 (d, 2 H, J = 1.2 Hz), 7.60 (dd, 2 H, J1 = 9.0 Hz, J2 = 1.2 Hz), 7.06 (d, 2 H, J = 5.4 Hz), 4.16 (s, 4 H), 3.82 (s, 4 H), 3.48 (s, 2 H), 1.92 (s, 2 H), 1.44 (s, 2 H), 1.35 (m, 23 H), 0.93 (t, 3 H, J = 6.6 Hz). 13 13C NMR (150 MHz, CD3OD): δ 155.9, 143.0, 139.8, 138.2, 127.4, 125.3, 118.7, 115.5, 98.9, 54.6, 51.1, 38.3, 31.5, 29.2, 29.2, 29.2, 29.2, 29.1, 29.0, 28.9, 28.7, 26.1, 23.0, 22.2, 12.9. ESI-HRMS: C 36 H 50 Cl2N5 + for m / z [M + H] + calculated value 622.3438, measured value 622.3505.

[0161] BAQ15. Yield: 290 mg, 45.5%. 1 1H NMR (600 MHz, CD3OD): δ 8.56 (d, 2 H, J = 9.0 Hz), 8.48 (d, 2 H, J = 6.0 Hz), 7.86 (d, 2 H, J = 1.2 Hz), 7.59 (dd, 2 H, J1 = 9.0 Hz, J2 = 1.2 Hz), 7.07 (d, 2 H, J = 6.0 Hz), 4.16 (s, 4 H), 3.82 (s, 4 H), 3.48 (s, 2 H), 1.92 (s, 2 H), 1.44 (s, 2 H), 1.35 (m, 25 H), 0.93 (t, 3 H, J = 6.6 Hz). 13C NMR (150 MHz, CD3OD): δ 155.8, 143.0, 139.7, 138.1, 127.3, 125.3, 118.7, 115.4, 98.9, 54.5, 51.0, 3 8.2, 31.5, 29.2, 29.2, 29.2, 29.1, 29.0, 28.9, 28.7, 26.1, 23.0, 22.2, 12.9. ESI-HRMS:C 37 H 52 Cl2N5 + m / z [M+H] + Calculated value: 636.3594, measured value: 636.3661.

[0162] BAQ16. Yield: 280 mg, 43.0%. 1 H NMR (600 MHz, CD3OD): δ 8.56 (d, 2 H, J=9.0 Hz), 8.48 (d, 2 H, J=6.6 Hz), 7.86 (d, 2 H, J=1.8 Hz), 7.59 (dd, 2 H, J1=9.0 Hz, J2=1.8 Hz), 7.07 (d, 2 H, J=7.2 Hz), 4.17 (m, 4 H), 3.84 (m, 4 H), 3.50 (t, 2 H, J=7.8 Hz), 1.92 (m, 2 H), 1.44 (m, 2 H), 1.30 (m, 27 H), 0.93 (t, 3 H, J=7.2 Hz). 13 C NMR (150 MHz, CD3OD): δ 155.9, 143.0, 139.7, 138.1, 127.3, 125.2, 118.7, 115.4, 98.8, 54.5, 51.0, 3 8.2, 31.5, 29.2, 29.2, 29.2, 29.1, 28.9, 28.9, 28.7, 26.1, 23.0, 22.1, 12.9. ESI-HRMS:C 38 H 54 Cl2N5 + m / z [M+H] + Calculated value: 650.3751, actual value: 650.3774.

[0163] BAQ18. Yield: 290 mg, 42.7%. 1H NMR (600 MHz, CD3OD): δ 8.56 (d, 2 H, J=9.0 Hz), 8.48 (d, 2 H, J=5.4 Hz), 7.86 (s, 2 H, J=1.8 Hz), 7.56 (d, 2 H, J1=8.4 Hz), 7.06 (d, 2 H, J=6.6 Hz), 4.16 (s, 4 H), 3.82 (m, 4 H), 3.48 (s, 2 H), 1.91 (s, 2 H), 1.43 (m, 2 H), 1.30 (m, 29 H), 0.93 (s, 3 H). 13 C NMR (150 MHz, CD3OD): δ 155.9, 143.0, 139.8, 138.2, 127.4, 125.2, 118.7, 115.5, 98.8, 54.5, 5 1.1, 38.2, 31.5, 29.2, 29.1, 28.9, 28.9, 28.7, 26.1, 23.0, 22.2, 12.9. ESI-HRMS:C 38 H 54 Cl2N5 + m / z [M+H] + Calculated value 650.3751, measured value 650.3774. ESI-HRMS:C 40 H 56 Cl2N5 + m / z [M+H] + Calculated value: 678.4064, measured value: 678.4069. [ka]

[0164] Synthesis of compound 1. Ethylene N-carboethoxyphthalimide (2.34 g, 10.7 mmol, 1.00 equiv) and triethylamine (1.49 mL, 10.7 mmol, 1.00 equiv) were added to a solution of 4,4-diethoxybutylamine (1.84 mL, 10.7 mmol, 1.00 equiv) in THF (30 mL). The reaction mixture was stirred at room temperature for 12 h. After removing the solvent under reduced pressure, the crude material was purified on a silica column eluted with 1:20 EtOAc:Hexane to give a clear oil. (2.9 g, 94.0 mmol, 93% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.87-7.83 (m, 4H), 4.47 (t, J = 5.4 Hz, 1H), 3.58-3.51 (m, 4H), 3.43-3.38 (m, 2H), 1.61-1.60 (m, 2H), 1.54-1.51 (m, 2H). ESI-HRMS m / z 314.1361 [M+Na] + . [ka]

[0165] Synthesis of compound 2. A solution of compound 1 (2.2 g, 7.6 mmol, 1.00 equiv.) in acetone (20 mL) and 1 M aqueous HCl (15.2 mL, 15.2 mmol, 2.00 equiv.) was refluxed (80° C.) and vigorously stirred for 1 h. Acetone was evaporated under reduced pressure and the resulting aqueous layer was extracted three times with Et2O. The combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by column chromatography eluting with 1:2 EtOAc:hexane to give a waxy white solid (1.2 g, 74% yield). 1 H NMR (600 MHz, DMSO-d6) δ 9.64 (t, J = 1.2 Hz, 1H, NH2), 7.88-7.83 (m, 4H), 3.61 (t, J = 7.2 Hz, 2H), 2.54-2.51 (m, 2H), 1.85-1.83 (m, 2H). [ka]

[0166] Synthesis of BAQ4q. Compound 2 (434 mg, 2 mmol, 2.0 equiv.) and acetic acid (10 μL) were added to a solution of BAQ (426.5 mg, 1.0 mmol, 1.00 equiv.) in methanol (40 mL) and stirred at room temperature for 30 min. Sodium cyanoborohydride (126 mg, 2 mmol, 2 equiv.) was slowly added and stirred for 24 h. The reaction solution was diluted with dichloromethane (150 mL), washed with saturated sodium carbonate, water, and brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the mixture was purified by silica chromatography eluting with 20:1 dichloromethane:methanol to give a white solid (520 mg, 83% yield). 1 H NMR (600 MHz, DMSO-d6) δ 8.27 (d, J = 5.4 Hz, 2H), 7.97 (d, J = 9.0 Hz, 2H), 7.78 (s, 4H), 7.67 (d, J = 2.4 Hz, 2H), 7.19 (dd, J1= 9.0 Hz, J2= 2.4 Hz, 2H), 7.09 (m, 2H), 6.39 (d, J = 6.0 Hz, 2H), 3.51 (t, J = 7.2 Hz, 2H), 3.31 (t, J = 6.0 Hz, 4H), 2.77 (t, J = 6.6 Hz, 4H), 2.58 (t, J = 6.6 Hz, 2H), 1.60-1.55 (m, 2H), 1.45-1.40 (m, 2H). ESI-HRMS 627.2035 [M+H] + . [ka]

[0167] Synthesis of BAQ4a. BAQ4q (314 mg, 0.5 mmol, 1.0 equiv.) was dissolved in ethanol, hydrazine (2.5 mmol, 5.0 equiv.) was added, and the reaction solution was stirred at reflux for 12 h. The reaction mixture was then allowed to warm to room temperature. After removing the precipitate by filtration, the filtrate was concentrated and then added to ether (100 mL) to form a white precipitate, which was collected as compound 5 (200 mg, 80% yield). 1 H NMR 8.30 (d, J = 5.4 Hz, 2H), 8.03 (d, J = 9.6 Hz, 2H), 7.78 (s, 4H), 7.71 (d, J = 2.4 Hz, 2H), 7.27 (dd, J1= 9.0 Hz, J2= 2.4 Hz, 2H), 7.05 (t, J =5.4 Hz, 2H), 6.40 (d, J = 5.4 Hz, 2H), 3.31 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 6.0 Hz, 4H), 2.78 (t, J = 7.2 Hz, 4H), 2.54 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.41-1.38 (m, 2H), 1.29-1.26 (m, 2H). . ESI-HRMS 497.1981 [M+H] + . [ka]

[0168] Synthesis of PBC. Pheophorbide a (296 mg, 0.5 mmol, 1.0 equiv), 6-chloro-1-hydroxybenzotriazole (102 mg, 0.6 mmol, 1.2 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (93 mg, 0.6 mmol, 1.2 equiv), and N,N-diisopropylethyleneamine (174 μL, 1.0 mmol, 2 equiv) were suspended in anhydrous dichloromethane (75 mL) and stirred at room temperature for 30 min. Compound 5 (314 mg, 0.5 mmol, 1.0 equiv) was added to the reaction mixture, which was then stirred for 48 h. The mixture was purified by silica chromatography eluting with 20:1 dichloromethane:methanol to give a black solid (200 mg, 37% yield). 1H NMR (600 MHz, CD3OD) δ 8.94 (s, 1H), 8.78 (s, 1H), 8.58 (s, 1H), 7.86 (dd, J1= 6.0 Hz, J2= 3.6 Hz, 2H), 7.86 (dd, J1= 6.0 Hz, J2= 3.6 Hz, 2H), 7.81 (dd, J1= 18.0 Hz, J2= 11.4 Hz, 2H), 7.63 (m, 1H), 7.54 (d, J = 6.0 Hz, 2H), 7.18 (dd, J1= 9.0 Hz, J2= 2.4 Hz, 1H), 6.88 (m, 2H), 6.55 (d, J=8.4Hz, 2H), 6.22 (dd, J1= 8.4 Hz, J2= 1.8 Hz, 2H), 6.17 (d, J = 18.0 Hz, 1H), 6.10 (d, J = 11.4 Hz, 1H), 5.55 (d, J = 18.0 Hz, 1H), 4.52-4.51 (m, 1H), 4.15-4.14 (m, 1H), 3.88 (s, 3H), 3.72-3.67 (m, 2H), 3.30 (s, 3H), 3.2 (q, J2= 7.2 Hz, 4H, トリエチルアミン), 2.89 (m, 1H), 2.80 (s, 3H), 2.75 (m, 1H), 2.63-2.61 (m, 2H), 2.56-2.53 (m, 1H), 2.50-2.43 (m, 4H), 2.3-2.16 (m, 8H), 1.94 (s, 1H), 1.82 (d, J = 7.2Hz, 3H), 1.47 (t, J = 7.8Hz, 3H), 1.35 (m, 10H+6H トリエチルアミン), 1.11 (m, 4H). ESI-HRMS 1071.4576 [M+H] + .

change

[0169] Synthesis of CAB. Cholic acid (204 mg, 0.5 mmol, 1.0 equiv), 6-chloro-1-hydroxybenzotriazole (102 mg, 0.6 mmol, 1.2 equiv), 1-ethylene-3-(3-dimethylaminopropyl)carbodiimide (93 mg, 0.6 mmol, 1.2 equiv), and N,N-diisopropylethylamine (174 μL, 1.0 mmol, 2 equiv) were suspended in anhydrous dichloromethane (75 mL) and stirred at room temperature for 30 min. BAQ4a (314 mg, 0.5 mmol, 1.0 equiv) was added to the reaction mixture, which was then stirred for 48 h. The mixture was purified by silica chromatography eluting with 20:1 dichloromethane:methanol to give a black solid (220 mg, 49.5% yield). ESI-HRMS 887.4774 [M+H] + . [ka]

[0170] Synthesis of BAQ5h. To a solution of BAQ (426.5 mg, 1.0 mmol, 1.00 equiv) in methanol (40 mL), glutaric dialdehyde (211 μL, 2.0 mmol, 2.00 equiv) and acetic acid (10 μL) were added and stirred at room temperature for 30 min. Sodium cyanoborohydride (126 mg, 2 mmol, 2 equiv) was slowly added and stirred for 24 h. The reaction solution was diluted with dichloromethane (150 mL), washed with saturated sodium carbonate, water, and brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the mixture was purified by silica chromatography eluting with 20:1 dichloromethane:methanol to give a white solid (520 mg, 83% yield). 1H NMR (600 MHz, CD3OD) δ 8.23 ​​(d, J = 5.4 Hz, 2H), 7.60-7.56 (m, 4H), 7.02 (dd, J1= 9.0 Hz, J2= 1.8 Hz, 2H), 6.44 (t, J = 5.4 Hz, 2H), 3.58 (t, J = 6.6 Hz, 2H), 3.42 (t, J = 5.4 Hz, 4H), 2.91 (t, J = 6.0 Hz, 4H), 2.75-2.72 (m, 2H), 1.66-1.57 (m, 4H), 1.51-1.47 (m, 2H). ESI-HRMS 512.1961 [M+H] + . [ka]

[0171] Synthesis of DCQO. 4,7-Dichloroquinoline (DCQ) (2 g, 10 mmol) was dissolved in 50 mL and stirred vigorously (500 rpm) on ice-water bath for 15 min. mCPBA (2.7 g, 12 mmol) was carefully added to the reaction solution in the bath (4 times). The resulting reaction mixture was stirred at room temperature for 12 h (500 rpm). TLC showed that the starting material was completely converted to one large spot. Dichloromethane (100 mL) and potassium carbonate (4.1 g, 30 mmol) were added to the reaction solution and stirred at room temperature for 1 h (300 rpm). The mixture was poured into a 500 mL beaker with 200 mL of water and stirred for an additional 1 h (300 rpm). (The organic phase was collected, washed with saturated sodium carbonate (75 mL×3), water (75 mL×3), and brine (75 mL×3), respectively, and dried over anhydrous sodium sulfate overnight. After filtration, the solvent was evaporated under reduced pressure, and the crude material obtained was recrystallized with 80 mL of acetonitrile. The solid product obtained was collected by filtration and then dried under vacuum to give 1.8 g of DCQO as a white solid. 1HNMR (600 MHz, CDCl3) δ 8.79 (d, J=1.8 Hz, 2 H), 8.44 (d, J=6.6 Hz, 2 H), 8.44 (d, J=8.4 Hz, 2 H), 7.71 (d, J1=9.0 Hz, J2=2.4 Hz, 2 H), 7.38 (d, J=6.0Hz, 2H). HRMS (ESI):C9H6Cl2NO [M+H] + Calculated m / z for 213.9821, measured m / z 213.9834. [ka]

[0172] Synthesis of BAQO. To a solution of DCQO (2.14 g, 10 mmol) in 30 mL of absolute ethanol, sodium bicarbonate (840 mg, 10 mmol) and diethylenetriamine (432 μL, 4 mmol) were added. The mixture was refluxed at 95 °C for 48 h. TLC was used to show the formation of the target material (TM, yellow spot). Ethanol was evaporated under reduced pressure, and the residue was resuspended in 30 mL of methanol, which was slowly dripped into a 300 mL beaker with a mixture of 100 mL of hydrochloric acid (1 M) and 50 mL of dichloromethane. The aqueous phase was collected, washed with dichloromethane (50 mL × 2), and alkalized to pH 10 with 10 M NaOH (12 mL), resulting in the formation of a yellow precipitate. The precipitate was collected, washed with water (30 mL × 3), and dried under vacuum to give 850 mg of BAQO as a yellow solid. 1 HNMR (600 MHz, CD3OD) δ 8.51 (d, J=1.8 Hz, 2H), 8.35 (d, J=7.2 Hz, 2H), 8.14 (d, J=9.0 Hz, 2H), 7.55 (d, J1=9.0 Hz, J2=2.4 Hz, 2H), 6.63 (d, HRMS (ESI):C 22 H 22 Cl2N5O2[M+H]+ Calculated m / z for 458.1145, measured 458.1126. [ka]

[0173] Synthesis of BAQ12O. A mixture of BAQO (916 mg, 2 mmol), dodecylaldehyde (1.8 mL, 8 mmol), and acetic acid (20 μL) was stirred vigorously (500 rpm) at room temperature for 30 min. Sodium cyanoborohydride (377 mg, 6 mmol) was then added slowly. The reaction mixture was stirred at room temperature for another 12 h. TLC showed that the starting material was completely converted into one large spot. After concentrating the solvent to 25 mL, the resulting residue was diluted with 75 ml of dichloromethane). The organic phase was washed three times with 100 mL of saturated sodium bicarbonate. The emulsion layer was collected and then filtered to give a yellow solid, which was washed with water (30 mL × 3) and ethylene ether (30 mL × 3). The collected yellow solid was dried under vacuum to give 1.2 g of BAQ12O. 1 HNMR (CD3OD, 600MHz) δ 8.43 (d, J=1.8 Hz, 2H), 8.31 (d, J=7.2 Hz, 2H), 7.82 (d, J=9.0 Hz, 2H), 7.31 (d, J1=9.0 Hz, J2=2.4 Hz, 2H), 6.56 (d, J=7.2 Hz, 2H), 3.50 (t, J=6.0 Hz, 4H), 2.94 (t, J=6.0 Hz, 4H), 2.71 (t, J=7.2 Hz, 2H), 1.55 (m, 2H), 1.32 (m, 18H), 0.92 (t, J1=6.6 Hz, 3H). CNMR (CD3OD, 150MHz) δ 148.0,140.1,139.9,138.4,127.6,123.9,118.7,117.9,98.09,54.9,52.3,41.5,32.4,30.2,30.1,30.1,29.8,28.1,27.7,23.1,13.8. HRMS (ESI):C 34 H 46 Cl2 Calculated m / z for N5O2[M+H]+ 626.3023, found 626.3060. [ka]

[0174] Synthesis of BAQ1O. This compound was prepared using the method of BAQ12O. Formaldehyde was used as the starting material. ESI-HRMS 472.1325 [M+H] + . [ka]

[0175] Synthesis of BAQAO. BAQO (229 mg, 0.5 mmol) in 5 mL of acetic anhydride was refluxed for 6 h. Excess acetic anhydride was removed under reduced pressure. The residue was taken up in cold diethyl ether to give the product as a yellow solid (180 mg). ESI-HRMS 500.1246 [M+H] + . [ka]

[0176] Synthesis of BAQ5hO. This compound was prepared using the method of BAQ5h. BAQO was used as the starting material. ESI-HRMS 544.1870 [M+H] + . [ka]

[0177] Synthesis of BAQ4qO. This compound was prepared using the method of BAQ4q. BAQO was used as the starting material. ESI-HRMS 659.1946 [M+H] + . [ka]

[0178] Synthesis of BAQ4aO. This compound was prepared using the method of BAQ4a. BAQ4qO was used as the starting material. ESI-HRMS 529.1884 [M+H] + . [ka]

[0179] Synthesis of BAQ13O. This compound was prepared using the method of BAQ12O. Tridecanal was used as the starting material. 1 HNMR (800 MHz, DMSO-d6) δ 9.79 (s, 2 H), 8.91 (d, J=7.2 Hz, 2H), 8.78 (d, J=9.6 Hz, 2 H), 8.16 (d, J=1.6 Hz, 2H), 7.80 (dd, J1=9.6 Hz, J1=1.8 Hz, 2H), 6.97 (d, J=8.0 Hz, 2H), 4.04 (s, 4H), 3.68-3.62 (m, 6 H), 1.70 (s, 2 H), 1.27-1.17 (m, 21H), 0.87 (t, J=7.2 Hz, 3H).

[0180] Synthesis of BAQ14O. This compound was prepared using the method of BAQ12O. Tetradecanal was used as the starting material. 1 HNMR (800 MHz, DMSO-d6) δ 9.62 (s, 2 H), 8.88 (d, J=8.0Hz, 2H), 8.70 (d, J=9.6Hz, 2 H), 8.15 (d, J=2.4 Hz, 2H), 7.77 (dd, J1=8.8 Hz, J1=1.6 Hz, 2H), 6.95 (d, J=7.2 Hz, 2H), 4.02 (s, 4H), 3.68-3.62 (m, 6 H), 1.68 (s, 2 H), 1.27-1.15 (m, 23H), 0.86 (t, J=5.4 Hz, 3H).

[0181] Synthesis of BAQ15O. ​​This compound was prepared using the method of BAQ12O. Pentadecanal was used as the starting material.1 HNMR (800 MHz, DMSO-d6) δ 9.63 (s, 2 H), 8.88 (d, J=8.0 Hz, 2H), 8.70 (d, J=8.8 Hz, 2 H), 8.15 (d, J=2.4 Hz, 2H), 7.77 (dd, J1=8.8 Hz, J1=1.6 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 4.01 (s, 4H), 3.68-3.62 (m, 6H), 1.68 (s, 2H), 1.28-1.15 (m, 25H), 0.86 (t, J=5.4 Hz, 3H).

[0182] Synthesis of BAQ16O and BAQ18O. The compounds were prepared using the method of BAQ12O. Hexadecanal was used as the starting material for BAQ16O, and octadecanal was used as the starting material for BAQ18O.

[0183] Additional BAQO derivatives can be prepared using the methods of BAQ12O using the appropriate aldehyde and ketone starting materials. Example 2: Nanocarriers

[0184] Preparation and characterization of BAQ ONNs. NPs were prepared through reprecipitation method. Methanol containing BAQ derivatives was added dropwise to MilliQ water (volume ratio, 1:10) with stirring for 5 min, and homogeneous NPs were obtained after rotary evaporation (40 °C, 20 min), which were then characterized using a Zetasizer Nano ZS (Malvern). TEM samples were prepared by dropping 0.5 mM NPs onto a carbon square mesh and air-dried, and then observed on a Talos L120C TEM (FEI) at an accelerating voltage of 80 kV. To determine the drug content in the nanoformulations, the prepared drug-loaded NPs were cut by a centrifugal filter (ultracel-10 kDa, Millipore), and the absorbance of the filtrate (diluted with DMSO, 1:10, volume ratio) was measured to calculate the drug concentration.

[0185] Discovery of BAQ derivatives as potential ONNs. BAQ12–BAQ18 were designed by hybridization of key structural elements of the lysosomotropic autophagy inhibitor Lys05 with the lysosomotropic detergent MSDH to achieve pharmacological fusion (Figure 1). Based on the self-assembly principle, it was predicted that the inclusion of a long hydrophobic tail with a cationic BAQ head would facilitate the formation of nanoparticles (NPs). Since the BAQ head has a calculated pKa of 8.4, this self-assembly should be dependent on the surrounding pH, with NPs formed under neutral conditions and dissociating into free building blocks after protonation in an acidic environment.

[0186] Compounds (BAQ12 to BAQ18) were synthesized. 1 H NMR, 13The structures were confirmed by C NMR and HRMS spectra (Figure 7). Unlike Lys05 and MSDH, BAQ12–BAQ18 were not completely soluble in water in the form of free base or hydrochloride. However, the lipophilic cations enabled spontaneous self-assembly through nanoprecipitation in water, resulting in homogeneous and opaque NP solutions. The aggregate NPs of BAQ12–BAQ18 had similar nanoscale characteristics, including their size (100–140 nm), polydispersity index (PDI) values ​​less than 0.1, and positive surface charge (approximately +40 mV) (Table 1 and Figure 8A). The pH-responsive dissociation behavior was then evaluated by monitoring the size change of the particles (Figure 2A). All BAQ NPs remained intact under near-neutral conditions and dissociated only under relatively acidic conditions. The critical dissociation pH was 5.5–6.0 for BAQ12–BAQ14 and 5.0–5.5 for BAQ15–BAQ18. Upon protonation in an acidic environment, the BAQ12–BAQ18 lipophilic cations became amphiphilic molecules and acquired surface activity. Hemolysis tests were then used to evaluate the pH-responsive biomembrane disruption ability of the compounds. None of the NPs had a hemolytic effect under nearly neutral conditions (pH ≥ 6.5), but induction of hemolysis began when the pH was below 6.0 (Figures 2B and 8B). Among the compounds, BAQ12 NPs and BAQ13 NPs showed the strongest hemolytic activity, inducing up to 90% hemolysis under simulated lysosomal conditions (pH 4.0–5.5); in contrast, BAQ14 NPs induced moderate hemolysis (70%), and only BAQ15–BAQ18 NPs caused about 50% hemolysis. In controls, the conventional lysosomal detergent MSDH showed only a weak hemolytic response to pH, and detergent-free Lys05 induced no observable hemolysis across the pH range at the same concentrations.Since LMPs are potential stimuli for apoptosis, BAQ12 and BAQ13, whose detergents can be activated in liposomes, may be effective in directly inducing cancer cell death.Moreover, when titrated with hydrochloride (HCl), BAQ12 NPs and BAQ13 NPs showed a clear plateau of pH within a narrow pH range (at approximately pH 6.0), indicating their strong pH buffering ability (Figure 2C). In contrast, the pH values ​​of the other NPs (BAQ14-BAQ18) decreased proportionally, and only short pH plateaus were observed for Lys05 (pH 7.2) and MSDH (pH 6.2). Since sufficient acidification is necessary for lysosomal degradation, strong HCl is required. + BAQ12 and BAQ13, which have buffering capacity, showed greater potential than other compounds in inducing lysosomal dysfunction, and therefore BAQ12 and BAQ13 may attenuate tumor cell proliferation. [Table 1]

[0187] To verify the therapeutic efficacy of BAQ12-BAQ18, preliminary screening was performed using MTS assay in various cancer cell lines. These derivatives showed different levels of antiproliferative effects within 24 hours of treatment. BAQ12 and BAQ13 were highly effective, showing approximately 3-fold, 20-fold, and 10-fold higher potency than Lys05, HCQ, and MSDH, respectively, but the activity steadily decreased as the hydrophobic tail was extended from 14 to 18 carbons (Table 1 and Figure 8C). This decrease was due to the cleaning ability and H + This was attributed to a gradual decrease in buffering capacity. Based on the above results, BAQ12 and BAQ13 were selected as representatives for constructing BAQ ONN in the following studies.

[0188] pH-responsive assembly and large drug loading capacity. The dissociation phase transition of the pH-responsive assembly of BAQ ONNs was then revealed by transmission electron microscopy (TEM). At pH 7.4, the NPs exhibited a strong Tyndall effect and liposome-like nanostructures with a diameter of about 100 nm and a bilayer thickness of about 5 nm (Figure 2D). These results were consistent with the dynamic light scattering (DLS) measurements. In contrast, at pH 5.0, the solution lost its Tyndall effect and vesicles were absent under TEM, demonstrating that the NPs dissociated under this condition (Figure 2E). We then investigated the BAQ ONN release behavior at physiological pH (7.4) and lysosomal pH (5.0). As shown in Figure 2F, BAQ12 NPs and BAQ13 NPs were almost completely released (about 90%) over 8 h at pH 5.0, whereas only about 10% of the drug was released over 24 h under neutral conditions. Considering that lysosomes maintain a pH range of 4.0–5.5, it is conceivable that upon reaching these compartments, BAQ ONNs would dissociate into free small molecules and thus exert their therapeutic effects. The critical aggregation concentrations (CAC) of BAQ12 NPs and BAQ13 NPs were measured to be 0.76 μM and 0.25 μM, respectively (Figure 2G). The three-fold difference observed between them indicated that BAQ13 could form NPs more easily than BAQ12, despite the difference between their molecular structures being only one methylene unit. The two NPs also showed sufficient stability of particle size over a relatively long period of time at room temperature, even in the presence of 10% serum or 0.5 mM bovine serum albumin (Figures 9A–9F). In addition, BAQ13 NPs showed greater stability than BAQ12 NPs during such long-term storage. This is likely due to the difference in CAC between the two.

[0189] The next study was whether the liposome-like BAQ ONNs could encapsulate additional drugs. Upon nanoprecipitation of BAQ13 and various drugs, uniform NPs with a monomodal size distribution were spontaneously formed (Table 2 and Figure 9G). BAQ13 NPs showed large drug loading (up to 50%, mass ratio) as well as a drug encapsulation efficiency of about 90% (Table 2). This indicated that BAQ13 NPs could overcome the drug loading constraints of traditional liposome-based and polymer-based drug delivery systems. It is very encouraging that these single NPs consisting of a single small molecule therapeutic exhibit such a strong drug loading ability. [Table 2]

[0190] Accumulation in lysosomes and lysosomal disruption. To verify the accumulation of BAQ ONN in lysosomes, a near-infrared fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD), was loaded for labeling and tracking. As expected, lysosomal puncta (green) in MIA PaCa-2 cells stained with Dextran-Alexa Fluor 488 (AF488) closely overlapped with DiD-labeled NPs (red). This suggests that BAQ ONN was rapidly taken up by cells and accumulated in lysosomes (Figure 3A and Figure 10A). When this accumulation occurred, BAQ ONN reduced LysoTracker-positive puncta, indicating the ability of BAQ ONN to deoxygenate lysosomes as well as Lys05 and MSDH (Figure 3B and Figure 10B-10C).

[0191] The induction of LMP by BAQ12 and BAQ13 was examined by staining live cells with the dye acridine orange (AO). Compared to cells treated with Lys05 and MSDH, cells treated with BAQ12 NPs or BAQ13 NPs showed a decreased number of red puncta and an increased ratio of green to red fluorescence, suggesting that BAQ ONNs have an increased ability to induce lysosomal disruption in cancer cells (Figure 3C and Figures 10D-10E). This LMP effect was further confirmed by detecting the release of Dextran-AF488 from lysosomes. As shown in Figure 3D, treatment with BAQ ONNs resulted in a scattered staining pattern throughout the cytoplasm (indicating lysosomal leakage), while the fluorescence in control cells appeared restricted to punctate structures (representing intact lysosomes). BAQ ONNs were demonstrated to induce the release of cathepsin B, one important trigger of apoptosis, from isolated lysosomes through their LMP function (Figure 3E). Because LMP was not observed in cells treated with MSDH, the results suggested that BAQ12 and BAQ13 represent the next generation of lysosomotropic detergents.

[0192] Autophagy inhibition. To investigate the effect of BAQ ONN on autophagy, we measured the levels of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (SQSTM1) / p62 proteins, which are often used to monitor changes in the autophagy process. During autophagy, the cytosolic form of LC3 (LC3-I) is converted to a lipid-modified form (LC3-II), which is then recruited to the autophagosomal membrane. Meanwhile, the autophagy substrate SQSTM1 / p62 protein is degraded through selective incorporation into autophagosomes. Therefore, increased levels of both LC3-II and SQSTM1 / p62 should be observed upon inhibition of autophagy, whereas increased levels of LC3-II and decreased levels of SQSTM1 / p62 should be observed when autophagy is activated. As shown in Figures 3F-3G, compared with untreated and Lys05-treated cells, MIA PaCa-2 cells treated with BAQ ONN exhibited a significant concentration-dependent increase in the levels of both LC3B-II and SQSTM1 / p62 proteins. Such an increase was also observed after treatment with bafilomycin A1 (BfA1), a known autophagy inhibitor. These findings indicate that BAQ ONN can inhibit cellular autophagy more effectively than Lys05.

[0193] The autophagy inhibitory effect was then confirmed using LC3B-GFP imaging, since the formation of fluorescent LC3-II puncta in cells can be used to visualize the accumulation of autophagosomes. Cells treated with BAQ ONN produced prominent LC3B-GFP puncta in a concentration-dependent manner (Figure 3H and Figure S10F). Quantification of LC3B-GFP puncta per cell revealed that the autophagy inhibitory potency of BAQ ONN was greater than that of Lys05 (Figure 3I). To further verify, TEM was used to monitor the changes in the ultrastructural morphology of cells. As expected, compared with Lys05 and MSDH, BAQ ONN induced the formation of larger autophagic vesicles (AV) or autophagosomes in cells, which further confirmed the improved autophagy inhibitory effect of BAQ ONN (Figure 3J-3K). Taken together, these findings indicate that BAQ12 NPs and BAQ13 NPs outperformed the parent Lys05 in inhibiting autophagy; therefore, BAQ ONNs represent the generation of nanoformulated autophagy inhibitors.

[0194] Proton sponge effect and lysosomal dysfunction. As cationic molecules, both BAQ12 and BAQ13 have strong H +It has buffering capacity (Figure 2C). The above TEM results showed that BAQ ONN could significantly enlarge lysosomes (Figure 3J-3K), which demonstrates the proton sponge effect of BAQ ONN. To further explore these effects, the changes in the transcriptome of MIA PaCa-2 cells after treatment were characterized using RNA sequencing (RNA-seq). A total of 13,234 genes were examined and their expression levels were compared between the vehicle group, the Lys05 group, and the BAQ13 group. Using volcano plot analysis, 165 differentially expressed genes (DEGs) (fold change ≥ 2 and p-value ≤ 0.05) were found in the Lys05 group compared to the vehicle group, including 62 up-regulated genes and 103 down-regulated genes. In comparison, 390 DEGs were found in BAQ13-treated cells, including 209 upregulated and 181 downregulated genes (Figure 11A). Gene set enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that BAQ13 NPs induced robust upregulation of lysosome-related genes, such as V-ATPase, Cl-channel, protease, and lysosome-associated membrane protein (LAMP) genes (Figures 4A-4C and 11B). qPCR analysis also confirmed the upregulation of V-ATPase and Cl-channel genes, which notably indicated that BAQ ONNs possessed strong proton sponge properties (Figures 4D-4E).

[0195] The upregulation of key lysosomal enzyme genes (e.g., cathepsin and NEU1) also clarified the lysosomal dysfunction caused by BAQ ONN (Figure 4B-4C). This indicates the function of BAQ ONN in lysosomal disruption, as LAMP genes, which are believed to be partially responsible for maintaining lysosomal integrity, were also upregulated (Figure 11C). The BAQ ONN-treated group showed higher transcriptome levels of pro-apoptotic genes (BAX, BAK1, BAD, BIM, and PUMA), thus revealing an enhanced pro-apoptotic effect (Figure 11C-11D). The lysosomal dysfunction induced by BAQ ONN was then confirmed using lipidomics analysis. BAQ13 NP induced the accumulation of the acid sphingomyelinase (ASM) precursor sphingomyelin (SM), leading to a decrease in the level of its product, ceramide (Cer) (Figure 11E). In addition, the levels of phospholipase A (PLA) precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PI), were decreased, whereas the levels of their corresponding products, lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPI), were increased (Figure 6F).

[0196] The nanocarriers of the present invention include R 1 The nanoparticles can also include a complex in which the nucleoside is pheophorbide-a, forming a pheophorbide-a bisaminoquinoline conjugate (PBC). PBC nanoparticles are lysosome-targeted nanoassemblies with alterable morphology. PBC nanoparticles have a liposome-like morphology under physiological conditions and could transform into nanofibers after accumulation in lysosomes. The nanofibers formed in lysosomes can cause lysosomal dysfunction and initiate apoptosis in cancer cells. The nanoparticles also support highly effective lysosome-based photodynamic therapy, which can essentially overcome autophagy-associated drug resistance, because they contain photosensitizing groups in their structure. Example 3: In vitro studies

[0197] Cell lines and cell culture. Human pancreatic cancer cell lines (MIA PaCa-2, BXPC3, and PANC-1) were originally purchased from ATCC and kindly provided by the laboratory of Dr. Shiro Urayama. HT29, HCT116, H460, MCF7, NIH / 3T3, and IMR-90 cell lines were purchased from ATCC. Bone marrow cells were harvested from leg bone marrow of FVB / N mice. All cells were cultured in 10% fetal bovine serum, 100 μg mL ... -1 of penicillin, and 100 units mL -1 The cells were cultured in the corresponding medium supplemented with 100 mL of streptomycin in a humidified atmosphere of 5% CO2 / 95% air at 37°C. All cell lines were routinely checked for mycoplasma contamination.

[0198] Establishment of patient-derived pancreatic cancer stem cells (PCSCs). Patient pancreatic tissue was donated by Dr. Shiro Urayama's laboratory at UC Davis Medical Center. Patient consent was obtained using "Remnant Clinical Biospecimens" following the Institutional Review Board (UC Davis IRB Protocol #244896). Patient tumor tissue was harvested using collagenase IV and dispase (Stem Cell Technologies, Vancouver, Canada) and deformed by passing through a 70 μm filter. Cells were isolated using a BD FACSAria II cell sorter by labeling with the following antibodies (Biolegend, CA, USA): anti-CD44 (IM7, Cat: #338823), anti-CD326 (9C4, Cat: #324207), and anti-CD24 (ML5, Cat: #311119) (Figure 16). Purified PCSCs were harvested, maintained in Essential 8 Flex medium (Thermo Fischer), and trypsinized using Gentle Cell Dissociation reagent (Stem Cell Technologies, Vancouver, Canada). In the tumorsphere formation assay, a cold single-cell suspension of PCSCs in Essential 8 Flex medium was mixed with cold Matrigel (1:1, volume ratio), and then dropped (100 μL) slowly and evenly into the center of wells on a 24-well plate (5,000 cells per well). The Matrigel was left to solidify for 45–60 min at 37°C in a humidified incubator, and then warm medium (500 μL) was added to each well. Tumorspheres were formed in 2 weeks. To determine their tumorigenicity in vivo, PCSCs were counted and resuspended in a mixture of PBS and Matrigel (1:1), and then injected subcutaneously into the flanks of NRG mice.

[0199] Cell viability, cell proliferation, and colony formation. Cell viability was examined by MTS assay. Briefly, cells in 96-well plates (4,000 cells per well) were treated as indicated and then incubated with MTS reagent for 4 h. OD values ​​(490 nm) were determined by a microplate reader. The results were calculated using the formula [(OD treat -OD blank ) / (OD control -OD blank ) × 100%]. Drug combination data were analyzed by Combenefit 2.02. For cell proliferation assays, cells (50,000 cells per well) in 6-well plates were treated as indicated and counted manually every 24 h. Colony formation assays were also performed in 6-well plates at an initial density of 1,000-2,000 cells per well. After incubation for 10-20 days as indicated, cells were washed with PBS and stained with a solution of crystal violet and methanol for 20 min.

[0200] Apoptosis and caspase-3 / 7 activity. Cell apoptosis was measured using a FITC-Annexin V / PI apoptosis kit (AnaSpec). Briefly, treated cells were stained according to the manufacturer's instructions and detected on a BD FACSCanto II flow cytometer. Data were analyzed by FlowJo 7.6.1. For caspase-3 / 7 activity assay, cells (10,000 cells per well) in 96-well plates were treated as indicated, followed by addition of AMC caspase-3 / 7 assay kit (50 μL per well, AnaSpec). Fluorescence intensity (λ ex = 356 nm, λ em = 442 nm) was recorded by a microplate reader.

[0201] Cellular uptake and deoxygenation. To determine cellular uptake, lysosomes were incubated with Alexa Fluor 488-dextran (10 kDa, 100 μg mL-1 Cells were labeled with DiD-loaded BAQ NPs (10 uM, 1:10, mass ratio) for 36 h, followed by incubation with DiD-loaded BAQ NPs (10 uM, 1:10, mass ratio) for 2 h. For lysosomal deoxidation assay, cells were treated for 2 h and incubated with LysoTracker Red (100 nM, Thermo Fisher) for 1 h. Images of cells were captured using a Zeiss confocal microscope and analyzed by Zen 2.3 and ImageJ 1.51s.

[0202] Lysosomal integrity. Lysosomal integrity was measured in live cells by using AO (Thermo Fisher) or Alexa Fluor 488-dextran (10 kDa) staining. For AO staining, treated cells were incubated with AO (2 μg mL -1 ) for 1 h. For dextran staining, the dextran-loaded cells were treated for 12 h. Images were captured under a Zeiss confocal microscope and analyzed by Zen 2.3 and ImageJ 1.51s.

[0203] LC3B-GFP imaging. Cells in 96-well plates (5,000 cells per well) were transfected with the autophagy sensor LC3B-GFP (Thermo Fisher) for 12 h. After 4 h of treatment as indicated, cells were visualized by fluorescence microscopy (Olympus). Puncta per well were quantified using ImageJ 1.51s.

[0204] Lysosome isolation and cathepsin release. Lysosomes were isolated using a lysosome enrichment kit (Thermo Fisher) according to the manufacturer's protocol. Equal amounts of isolated lysosomes were incubated at 37°C for 12 h as indicated and then pelleted to intact lysosomes by centrifugation at 15,000 x g for 30 min at 4°C. Cathepsin B release into the supernatant was determined after incubation with 200 μM fluorescent live cathepsin B substrate III (Z-Arg-Arg-AMC) for 2 h (Ex=380 mm, Em=460 mm).

[0205] Hemolysis. PBS (10 mM, pH 7.4) containing red blood cells (2%) was incubated with NPs for 4 h at 37 °C. After centrifugation at 500 × g for 5 min, the extent of hemolysis was revealed by spectrophotometric determination of the amount of hemoglobin in the supernatant (540 nm). The hemolysis assay was used to evaluate the pH-dependent cleaning ability and toxicity of NPs.

[0206] Western blot. Cell or tumor samples were lysed with RIPA buffer (Thermo Fisher). After centrifugation (15 min, 12,000×g) at 4° C., protein concentrations in the supernatants were determined by Bradford protein assay dye (Bio-Rad). Immunoblotting was routinely performed and developed using a ChemiDoc™ MP imaging system.

[0207] TEM of cells and tumor tissue. MIA PaCa-2 cells (30,000 cells per well, Lab-Tek) in 8-well slide plates were treated as indicated for 48 h. Freshly harvested tumors were cut into 1 mm 3 The samples were fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde + 2% paraformaldehyde, transferred onto a carbon square mesh, and then observed under a Talos L120C TEM.

[0208] RNA-seq. Total RNA was extracted from treated MIA PaCa-2 cells (5 μM, 24 h) by RNeasy mini kit (Qiagen, Germany). Samples were sent to UC Davis Comprehensive Cancer Center's Genomics Shared Resource (GSR) for RNA-Seq analysis. Standard RNA-Seq libraries were prepared from 100 ng total RNA using NEBNext Ultra Directional RNA Library Preparation Kit (New England BioLabs). Libraries were then pooled and analyzed on an Illumina HiSeq 4000 system (2 × 150 bp, paired-end, 20 × 10 per sample). 6 Multiplex sequencing was performed at 1000 bp (>1000 reads). Normalized gene read count data were analyzed using fold change and t-test. Differentially expressed genes (DEGs) were retrieved for signaling pathway enrichment with Funrich software 3.1.3. Gene sets were from the MSigDB database (Broad Institute). GSEA was performed using GSEA version 3.0 within the online KEGG gene set categories with the following parameters: n = 1,000 permutations, and adjusted p < 0.05 and FDR < 0.05 were considered significant.

[0209] qPCR. Total RNA was isolated using TRIZOL reagent (Invitrogen) and phenol-chloroform extraction. cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) with 2 μg of total RNA in a 20 μL reaction. The resulting cDNA was diluted 1:20 in nuclease-free water and 4 μL was used in triplicate per qPCR reaction. qPCR was performed using Power SYBR Green PCR master mix (Thermo Fisher) on a CFX96 Real-Time PCR Detection System (Bio-Rad) including a no-template negative control. Amplification of GAPDH was used to normalize the levels of mRNA expression. Primer sequences are listed in Table 3. [Table 3]

[0210] Lipidomics. MIA PaCa-2 cells were treated with compounds (2.5 μM) for 48 h and samples were routinely prepared by harvesting 1.5 million cells from each group for RPLC-QTOF analysis. Samples were analyzed on a Vanquish UHPLC system, followed by data acquisition using a Q-Exactive HF hybrid quadrupole-Orbitrap mass analyzer. LC-MS data were processed using MS-DIAL 3.70. Statistical analysis was performed by first normalizing the data using a sum of known values ​​or mTIC normalizing and scaling each sample. R 3.5.1 was then performed on the normalized peak heights for statistical analysis. ANOVA analysis was performed with FDR correction and post-hoc testing.

[0211] In vitro antitumor activity of BAQ derivatives. To systematically investigate the in vitro antitumor effects, three pancreatic cancer cell lines (MIA PaCa-2, BxPC-3, and PANC-1) and two colon cancer cell lines (HT29 and HCT116) were selected for 48 h MTS assay. BAQ ONNs showed IC values ​​of 1–3 μM. 50The results showed that BAQ12 and BAQ13 alone were more effective than Lys05, HCQ, and MSDH, respectively, because they showed a potent anti-cancer effect (Figure 4F, Figure S12, and Table 4). These results also showed that treatment with BAQ12 or BAQ13 alone was more effective than combination therapy with Lys05 and MSDH. This suggests that a remarkable pharmacodynamic synergistic effect occurred when the pharmacophores were fused. The results of cell proliferation and colony formation assays further demonstrate the inhibitory effect of BAQ ONN on tumor cells (Figure 4G-4H). This improved anticancer activity of BAQ ONN can be attributed to its multifunction in inducing LMP, lysosomal dysfunction, and autophagy inhibition in cancer cells; these effects are considered to be important triggers of apoptosis. To investigate the pro-apoptotic effect of BAQ ONN, the apoptotic signals in MIA PaCa-2 cells and HT29 cells were subsequently detected. Treatment with BAQ ONN significantly increased both the levels of caspase 3 / 7 activity and apoptosis (Figures 4I-4J). The control, Lys05, increased the apoptotic signal in a concentration-dependent manner, whereas IC 50 Its effect at high concentrations close to 1000 mg / kg / day was still weaker than that of low concentrations of BAQ ONN. These results demonstrate that cancer cells are more susceptible to apoptosis when treated with multifunctional BAQ substances than when treated with Lys05, whose main function is autophagy inhibition. In addition, compared with the above panel of cancer cell lines, non-cancerous cell lines (including IMR-90 cells, NIH / 3T3 cells, and bone marrow cells) are relatively insensitive to BAQ ONN, thus indicating that the safety of these compounds is relatively high (Figure 12 and Table 4). [Table 4]

[0212] In vitro antitumor activity of BAQO derivatives. The in vitro antitumor effects of BAQO derivatives were investigated in pancreatic cancer stem cells (PCSCs). BAQ12O ONN had an IC of less than 5 μM. 50While the BAQO derivatives showed no toxicity up to a concentration of 100 μM (Figure 22B), the other BAQO derivatives showed no toxicity up to a concentration of 100 μM. These results indicate that the BAQO derivatives may be versatile as therapeutic or drug delivery agents without contributing to cytotoxicity. Example 4: In vivo studies

[0213] Animal model. To establish the subcutaneous xenograft model, 5 × 10 6 MIA PaCa-2 cells, 2 x 10 6 HT29 cells, or 2 x 10 4 PCSCs were suspended with Matrigel (Corning) and the PBS mixture (1:1, volume ratio) was subcutaneously injected into the right flank of nude or NRG mice, respectively.

[0214] Animal feeding. All animal experiments were performed according to a protocol (#20265) approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Female mice (4-6 weeks old) including BALB / c nude mice (Envigo), NRG mice (Jackson Laboratory), and FVB / N mice (Charles River) were purchased and housed in groups under standard conditions (22 ± 1°C, 50-60% humidity, 12-h light / 12-h dark cycle, free access to food and water).

[0215] In vivo treatment schedule. MIA PaCa-2 xenograft tumors (approximately 100 mm 3 NRG mice bearing 100-μg / mL IgG1 were randomly divided into five groups (n=6) and then injected iv every 3 days as indicated. For the HT29 xenograft model, six groups of 100 mm 3 Nude mice (n=6) bearing tumors of 1,000 mm were administered vehicle (saline, iv), Lys05 (ip), BAQ12 NPs (iv), BAQ13 NPs (iv), or irinotecan (ip) every 3 days. Treatment in the HT29 model was stopped on day 24, after which survival of mice in each group was recorded and 1,000 mm3 Mice bearing tumors larger than 100 μg / kg were considered dead. For co-delivery studies, NRG mice bearing PCSC tumors (n=5) were treated every 3 days with vehicle (saline, iv), napabucasin (iv), BAQ13 NPs (iv), BAQ13 NPs+napabucasin (iv and ip, respectively), and BAQ13 NPs@napabucasin (iv). Tumor volumes and body weights were recorded before each drug administration. Mice were euthanized when treatment was completed, and tumors were collected for further analysis.

[0216] In vivo toxicity study. The toxicity of BAQ NPs was investigated in female FVB / N mice by i.v. injection. Mice were administered various concentrations (10 mg kg -1 , 20 mg kg -1 , or 40 mg kg -1 Lys05, liposome@Lys05, BAQ12 NPs, and BAQ13 NPs were administered every 2 days. The condition of the mice was monitored daily, and the body weight was recorded every 2 days. Blood samples were collected and sent to the UCD Comparative Pathology Laboratory for complete blood count (CBC) and serum chemistry.

[0217] In vivo pharmacokinetic studies. Female Sprague-Dawley rats (200–250 g) were implanted with a catheter (Harland, Indianapolis, IN, USA) in the jugular vein for drug injection and blood withdrawal. Rats (n = 3) were injected with equivalent doses of DiD (0.5 mg kg -1 Free DiD containing BAQ12 NPs@DiD (10:1, mass ratio), and BAQ13 NPs@DiD (10:1, mass ratio) were injected, respectively. Blood samples were collected at the indicated time points and then centrifuged to obtain plasma. Plasma was diluted (1:100) with DMSO and the fluorescence intensity (λ Ex = 595 nm, λ Em = 665 nm) was measured using a microplate reader (SpectraMax M2).

[0218] In vivo / in vitro distribution. BAQ13 NP@DiD (10:1, mass ratio) was administered at 1.0 mg kg to nude mice bearing HT29 tumors. -1 The mice were administered BAQ13 NP@NAPA+DiD (10 / 2.5 / 1.0 mg kg−1) at 10-200 mg / kg doses. In vivo imaging studies were performed at the corresponding time points. Organs (brain, heart, lung, liver, spleen, kidney, intestine, and muscle) and tumors were harvested from the mice for ex vivo imaging. -1 The biodistribution of , iv) was investigated in NRG mice bearing PCSC tumors. Both in vivo and ex vivo imaging studies were performed as described above.

[0219] Statistics. Statistical analysis was performed using GraphPad Prism 7.0. Data are presented as mean ± SD, n = biological replicates or independent nanoparticle sample replicates. p values ​​were calculated using one-way ANOVA with Tukey's multiple comparison test or two-tailed Student's t test, as noted in each figure legend. ns., not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0220] Data availability. RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under the accession code GSE154323 [https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE154323].

[0221] Pharmacokinetics, biodistribution, and toxicity. The pharmacokinetics of BAQ ONN upon intravenous (i.v.) injection was studied in Sprague-Dawley rats. As shown in Figure 5A and Table 5, the serum concentration of BAQ ONN was higher than that of free DiD at the same time points up to 48 h, indicating that the plasma clearance of BAQ ONN was slower than that of DiD due to the nanoscale characteristics of BAQ ONN. DiD-labeled BAQ13 NPs were also used to investigate the biodistribution of NPs in nude mice bearing HT29 tumors. As expected, both in vivo and ex vivo imaging showed that the fluorescent signal of BAQ13 NPs was more clearly identifiable in the tumor area than in the surrounding normal tissues at 12 and 24 h after injection, indicating that BAQ ONN was tumor-targeted and biodistributed (Figures 5B-5C and Figures 13A-13B). This ability to target could be attributed to the relatively high ability to penetrate the tumor blood vessels, allowing passive accumulation of nanotherapeutics. The free DiD (control) group showed higher signals in the lung than in the tumor site (Figures 13A-13B). Notably, the tumor / lung ratio of fluorescence in the BAQ13 NPs group was approximately 4-fold greater than that in the free DiD group. These results demonstrated that BAQ ONNs have tumor-targeting biodistribution. [Table 5]

[0222] Next, a hemolysis assay was performed to evaluate the safety of BAQ ONN. Under physiological conditions, red blood cells were lysed at 0.25–1 mg mL, close to the working concentration used in animal therapeutic studies. -1 Treatment with Lys05, BAQ12 NPs, or BAQ13 NPs at concentrations of approximately 0.5 mg mL−1 (Figure 13C). Treatment with the control drug Lys05 resulted in a decrease in the number of NPs at concentrations of approximately 0.5 mg mL−1. -1 The hemolysis rate was significantly higher than that of treatment with BAQ12 NPs or BAQ13 NPs, indicating that the safety of BAQ ONN is greater than that of Lys05. In the following animal toxicity study in FVB / N mice, Lys05 treatment reduced the hemolysis rate at 10 mg kg after i.v.-1 It was found that even a low concentration of 40 mg kg caused sudden death in mice; in contrast, BAQ ONN treatment resulted in low mortality and no weight loss, demonstrating that BAQ ONN is safe when administered by injection (Figures 13D-13E). -1 In mice, BAQ13 NPs were better tolerated than BAQ12 NPs, with no mortality at all. This result can most likely be attributed to the high stability and low CAC of BAQ13 NPs. When liposomes were used to encapsulate Lys05 (liposome@Lys05), the formulation was found to be safe for i.v. injection (Figure 13E-13F). As autophagy plays an important role in intestinal homeostasis, intraperitoneal (i.p.) administration of BAQ12 NPs or BAQ13 NPs could result in increased autophagy inhibitory effects, causing intestinal disorders and weight loss in mice. H&E staining of tissue sections and hematological indices were examined using BAQ13 NPs administered at 20 mg kg IV via the tail vein. -1 This further suggests that iv administration of BAQ ONNs is well tolerated, as mice treated with BAQ NPs did not show any obvious abnormal changes (Figures 14A-14E). Therefore, BAQ ONNs should be administered by iv injection rather than ip injection to study their benefits in vivo.

[0223] Antitumor Efficacy as a Single Agent in Mice. After demonstrating the safety of BAQ ONN, the NPs were evaluated in a pancreatic xenograft model of MIA PaCa-2 cells for their anticancer efficacy. 3 NRG mice bearing 100 mg / kg IgG1-positive mice were randomly assigned to five groups (n = 6): saline (iv), Lys05 (ip), liposome@Lys05 (iv), BAQ12 NPs (iv), and BAQ13 NPs (iv). Mice were subsequently administered 20 mg / kg IgG1-positive mice every 3 days. -1The mice were treated with a dose of 0.01 mg / kg / day. The results in Figure 5D-5F show that treatment with BAQ12 NPs or BAQ13 NPs significantly slowed down tumor growth without interfering with body weight. This highlights the advantages of nanomedicine in drug delivery, since the control drug Lys05 showed no therapeutic effect under these conditions, whereas its nanoformulation, liposome@Lys05, induced increased tumor inhibition. It should also be emphasized that one-component formulations of self-assembling BAQ12 NPs or BAQ13 NPs were significantly more effective than either free Lys05 or nanoformulated Lys05. These findings clearly indicate the potential advantages of BAQ ONNs with regard to both drug discovery and drug delivery.

[0224] To further understand the in vivo effects of BAQ ONN, tumor tissues were collected for histological evaluation. Dramatic cell destruction, elevated levels of cleaved caspase-3, and decreased Ki67 expression were observed in both BAQ ONN groups, suggesting that tumors treated with BAQ ONN tended to die, become apoptotic, or dormant (Figure 5G-5H). LC3 expression was increased in both BAQ ONN groups (Figure 5H); this finding is one important clue to explain the in vivo autophagy inhibitory effects of both BAQ ONN. Subsequent immunoblot analysis further demonstrated that autophagy in tumors was blocked by BAQ ONN (Figure 5I). In addition to that, tissue ultrastructure was observed by TEM, and tumors treated with BAQ ONN were found to contain a greater number of large AVs than the tumor groups (Figure 5J). In the above assay, Lys05 nanoformulation, liposome@Lys05, also exhibited some effects that were not observed with vehicle or free Lys05. However, the effect of liposome@Lys05 was much weaker than that of either BAQ12 NPs or BAQ13 NPs.These tissue-level results revealed the superior autophagy inhibitory effect of BAQ ONNs in vivo.

[0225] The therapeutic efficacy of BAQ ONN in vivo was further demonstrated in another animal model consisting of mice bearing colon HT29 tumors. Compared with administration of vehicle or Lys05, administration of BAQ ONN significantly inhibited tumor growth (Figures 15A-15B). And BAQ13 NPs showed a better effect than BAQ12 NPs. Interestingly, this result was in contradiction with the results obtained in the in vitro amplification assay, where BAQ12 NPs were demonstrated to be more effective than BAQ13 NPs. This discrepancy could be explained by the difference in self-assembly behavior and pharmacokinetic profile between BAQ ONNs (Figures 2G, 5A). Moreover, BAQ13 NPs were also more effective at the reported therapeutic doses than FDA-approved irinotecan, whereas BAQ12 NPs showed similar efficacy to irinotecan. Survival analysis revealed that treatment with BAQ13 NPs resulted in a significantly longer survival time (median survival time 48 days) than the vehicle and irinotecan groups (median survival time 21 or 36 days, respectively) (Figure 15C and Table 6). The hybrid BAQ ONNs exhibit great potential for in vivo cancer treatment as single agents due to the integration of numerous advantages in terms of both pharmacodynamic effects and pharmacokinetic profiles. [Table 6]

[0226] Dual role of BAQ ONN in combination therapy. Although combination therapy based on autophagy inhibition is believed to sensitize tumors to conventional therapeutic agents, a current limitation is the insufficient effect of autophagy inhibitors. In addition, it is inconvenient that the drugs used in combination therapy have different pharmacokinetics and different dosing schedules. Because BAQ ONN has a 30-fold greater anticancer potency than HCQ and a greater capacity to encapsulate additional drugs, BAQ ONN may be able to simultaneously address these two challenges of pharmacodynamics and pharmacokinetics. To test this hypothesis, we used a pancreatic cancer stem cell (PCSC) line from patient-derived pancreatic adenocarcinoma tissue and established a xenograft model with large heterogeneity and a high proportion of tumor stroma (Figures 6A-6B and 16). In vitro results demonstrated that BAQ ONN has similar functions in terms of inhibition of lysosomes and autophagy in PCSCs and thus exhibits strong proapoptotic and antiproliferative activities (Figures 6C-6E and 17A-17C). For combination therapy, we selected napabucasin, a STAT3 inhibitor that can be encapsulated into BAQ13 NPs, because it can induce autophagy and synergize with BAQ13 NPs (Figure 6F and Figure 17D). Mice were randomly divided into five groups, including vehicle (saline), napabucasin, BAQ13 NPs, mixture (BAQ13 NPs + napabucasin), and BAQ13 NPs @ napabucasin (Figure 6G-6I). BAQ13 NPs moderately inhibited tumor growth, whereas napabucasin by itself did not show antitumor effects under these conditions. In vitro synergy between napabucasin and BAQ13 NPs was observed, although the mixture group did not show enhanced effects in vivo. This lack of in vivo effects was likely due to the small solubility and inefficient delivery of napabucasin. Nanoformulated napabucasin, when loaded into BAQ13 NPs (BAQ13 NPs@napabucasin), achieved satisfactory antitumor effects by synergizing with BAQ13 NPs. Significant changes in tumor histology were also observed in the BAQ13 NPs@napabucasin group, in which cells exhibited low proliferative activity (Figure 6J). In addition, none of the mice in the treatment groups showed obvious systemic toxicity (Figure 6I and Figure 17E).To further verify the ability of BAQ13 NPs to deliver napabucasin, another imaging study was performed on the PCSC model using DiD-labeled BAQ13 NPs@napabucasin. The results showed that the NPs obviously accumulated in the tumor site rather than in normal organs (Figures 6K-6L and 17F). These intriguing results demonstrate that BAQ13 NPs can function not only as a therapeutic agent but also as a delivery carrier in combination therapy; thus, BAQ13 NPs show promise for improving cancer treatment.

[0227] Antitumor Efficacy of BAQO Derivatives in Mice. BAQO derivatives can be used to form nanoparticles and for in vivo mouse studies. For example, BAQ12O NPs were used to treat mice bearing PCSC tumors. As shown in Figure 24A, mice treated with BAQ10O NPs had smaller tumor volumes, and there was a significant difference in tumor volume by day 27. Figure 24B shows that the tumor weight at the end of treatment with BAQ12O NPs was 50% less than that of the control. These interesting results demonstrate that BAQO derivatives can serve as promising agents for drug discovery and delivery.

[0228] Based on the ONN strategy and the principles of pharmacophore hybridization and molecular self-assembly, we developed self-delivering novel chemical entities, BAQ ONNs. These materials possessed improved properties for drug delivery and tumor-targeted biodistribution, as well as enhanced abilities to induce lysosomal disruption, lysosomal dysfunction, and autophagy block; thus, they exhibited significant anticancer effects. Surprisingly, single BAQ13 NPs were found to exhibit large drug-loading capacity and to be able to strongly synergize with and deliver additional drugs, making them promising for applications in combination therapy.

[0229] In contrast to traditional NPs, which typically have less than 20% active pharmaceutical ingredient (API) content and are difficult to synergize, BAQ ONNs have 100% API content, making them easier to synergize and scale up. BAQ ONNs are also superior to emerging one-component prodrug NPs because they are non-prodrug chemicals. All these advantages make it much easier to bridge BAQ ONNs to clinical trials. This is an important attempt to extend nanotechnology to the design of new chemical entities. The seamless connection between drug discovery and nanotechnology-assisted drug delivery will enable researchers to develop increasingly advanced nanomedicines with the advantages of cancer-targeted treatment and marketability.

[0230] Although the present invention has been described in some detail by way of illustrations and examples for clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be practiced within the scope of the appended claims. Additionally, each reference presented herein is incorporated by reference in its entirety as if each reference were individually incorporated by reference. In the event of a conflict between this application and a reference presented herein, this application shall control.

Claims

1. structure: 【Chemistry 1】 (wherein n is an integer from 1 to 7). or a pharma- ceutically acceptable salt thereof. 【Request 2】 【Chemistry 2】 2. The compound of claim 1, selected from the group consisting of: 【Request 3】 【Chemistry 3】 2. A compound selected from the group consisting of: or a pharma- ceutically acceptable salt thereof.

4. 4. A nanocarrier having an interior and an exterior, comprising a plurality of compounds according to any one of claims 1 to 3, or pharma- ceutically acceptable salts thereof, each compound self-assembles in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed on the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

5. The nanocarrier of claim 4, further comprising one or more hydrophobic drugs or imaging agents sequestered in the hydrophobic pocket.

6. The nanocarrier of claim 5 , wherein the hydrophobic drug is a chemotherapeutic agent, a molecular targeted agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof.

7. The nanocarrier of claim 6 , wherein the hydrophobic drug is an immunotherapeutic agent.

8. The nanocarrier of claim 6 , wherein the hydrophobic drug is a radiotherapeutic agent.

9. The nanocarrier of claim 6, wherein the hydrophobic drug is a chemotherapeutic agent or a molecular targeted agent.

10. The hydrophobic drug is selected from the group consisting of FLT-3 inhibitors, VEGFR inhibitors, EGFR TK inhibitors, Aurora kinase inhibitors, PIK-1 modulators, Bcl-2 inhibitors, HDAC inhibitors, c-MET inhibitors, PARP inhibitors, Cdk inhibitors, EGFR TK inhibitors, IGFR-TK inhibitors, anti-HGF antibodies, PI3 kinase inhibitors, AKT inhibitors, JAK / STAT inhibitors, inhibitors of checkpoint-1 or 2, focal adhesion kinase inhibitors, Map kinase kinase (mek) inhibitors, VEGF capture antibodies, everolimus, trabectedin, Abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, butabulin, ofatumumab, zanolibumab, edotecarin, tetrandrine, rubitecan, tesmilifen, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, Cilengitide, Dimatecan, IL13-PE38QQR; INO 1001, IPdR1 KRX-0402, Lucanton, LY 317615, Neurajiab, Vitespan, Rta 744, Sdx 102, Talampanel, Atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl l]-L-glutamic acid disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogens, bevacizumab, IMC-1C11, CHIR-258); 3-[5-(methylsulfonylpiperazinemethyl)-indolyl j-quinolone, vatalanib, AG-013736, AVE-0005, acetate of [D-Ser(Bu t) 6, Azgly 10] (Pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84 N 18 O 14 -(C 2 H 4 O 2 )X (where x = 1 to 2.4)], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, ionafarnib, BMS-214662, tipifarnib; amifosti , NVP-LAQ824, suberoylanilide hydroxamate, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, Epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozotocin Syn, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovastat, BMS-275291, squalamine,Endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin difitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab Cizumab (monoclonal antibody) and Erbitux, Cremophor-free paclitaxel, Epithilon B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zoledronate, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon alpha-2a, interferon alpha-2a, pegylated interferon alpha-2b, interferon alpha-2b, interferon alpha-2c, interferon alpha-2d, interferon alpha-2e, interferon alpha-2f, interferon alpha-2g, interferon alpha-2h ... -feron alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibrittonomab tiuxetan, androgen, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netupitant,The nanocarrier according to any one of claims 5 to 9, which is an NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspeg filgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib, or a combination thereof.

11. A nanocarrier according to any one of claims 4 to 7, comprising a plurality of compounds according to any one of claims 1 to 3.

12. A pharmaceutical composition for treating a disease, comprising the nanocarrier according to any one of claims 4 to 11.

13. 13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered in combination with one or more additional agents, wherein the additional agents are a chemotherapeutic agent, a molecular targeted agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof.

14. 14. The pharmaceutical composition of claim 13, wherein the additional pharmaceutical agent is an immunotherapeutic agent.

15. The pharmaceutical composition of claim 13, wherein the additional agent is a radiotherapeutic agent.

16. The pharmaceutical composition of claim 13, wherein the additional drug is a chemotherapeutic agent or a molecular targeted agent.

17. The additional agent is an FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, an inhibitor of checkpoint-1 or 2, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, butabulin, ofatumumab, zanolibumab, edotecarin, tetrandrine, rubitecan, tesmilifen, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, Cilengitide, Dimatecan, IL13-PE38QQR; INO 1001, IPdR1 KRX-0402, Lucanton, LY 317615, Neurajiab, Vitespan, Rta 744, Sdx 102, Talampanel, Atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl l]-L-glutamic acid disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogens, bevacizumab, IMC-1C11, CHIR-258); 3-[5-(methylsulfonylpiperazinemethyl)-indolyl j-quinolone, vatalanib, AG-013736, AVE-0005, acetate of [D-Ser(Bu t) 6, Azgly 10] (Pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 [C. 59 H 84 N 18 O 14 -(C 2 H 4 O 2 )X (where x = 1 to 2.4)], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, ionafarnib, BMS-214662, tipifarnib; amifosti , NVP-LAQ824, suberoylanilide hydroxamate, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, Epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozotocin Syn, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovastat, BMS-275291, squalamine,Endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin difitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab Cizumab (monoclonal antibody) and Erbitux, Cremophor-free paclitaxel, Epithilon B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zoledronate, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon alpha-2a, interferon alpha-2a, pegylated interferon alpha-2b, interferon alpha-2b, interferon alpha-2c, interferon alpha-2d, interferon alpha-2e, interferon alpha-2f, interferon alpha-2g, interferon alpha-2h ... -feron alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibrittonomab tiuxetan, androgen, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netupitant,The pharmaceutical composition according to any one of claims 13 to 16, which is an NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspeg filgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib, or a combination thereof.

18. The pharmaceutical composition of claim 12, wherein the disease is cancer.

19. 19. The pharmaceutical composition of claim 18, wherein the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and uterine cancer.

20. 13. The pharmaceutical composition of claim 12, wherein the disease is coronavirus, malaria, antiphospholipid syndrome, lupus, rheumatoid arthritis, chronic urticaria, or Sjogren's disease.

21. The pharmaceutical composition of any one of claims 12 to 20, which targets lysosomal disruption, lysosomal dysfunction, and / or autophagy inhibition.

22. 22. The pharmaceutical composition of claim 12 or 21, which targets the lysosome.

23. A pharmaceutical composition for imaging, comprising the nanocarrier according to any one of claims 4 to 11.

Images