Cyanine-Based Telodendrimers and Uses for Cancer Treatment - Patent application

Abstract

The present invention provides cyanine-modified telodendrimers, nanoparticles thereof, and methods of using the nanoparticles to treat various diseases, including cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 725,540, filed August 31, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] STATEMENT AS TO RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. 2R01CA115483-06, R01EB012569 and UJ01CA198880 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Near-infrared photothermal conversion agents (PTCAs) with strong and controllable optical properties have emerged as promising agents for biosensing, bioimaging and therapeutic applications. Light-triggered photothermal therapy (PTT) using PTCAs has shown several therapeutic advantages for localized cancer treatment. These include high spatial resolution, improved target selectivity, low to moderate adverse side effects, non-invasiveness without the need for surgery, rapid and effective treatment, negligible drug resistance and relatively low cost.

[0004] To date, many near-infrared PTCAs for PTT have been investigated in various tumor models using agents such as porphyrins, phthalocyanines, chlorin e6 (Ce6), gold-based nanomaterials, carbon nanotubes, and graphene oxide. Some conventional cyanine dyes have also been used as intermediates for light-mediated biomedical applications in the clinic. For example, indocyanine green (ICG), the only near-infrared agent approved by the US Food and Drug Administration (FDA), has been widely used clinically as an optical contrast to measure cardiac output, liver function, and blood flow, and in ocular angiography, highlighting the potential of using organic small molecule dyes for clinical translation and practical application. However, most of these PTCAs still face significant challenges in PTT clinical translation. These include poor biocompatibility (such as inorganic nanoagents), poor photothermal conversion efficiency, and inherently poor stability (rapid clearance from the kidney or liver in vivo, and poor resistance to photobleaching, such as small molecule dyes). The most important prerequisite for improving the photothermal conversion efficiency is to increase the light absorption in the target tissue and limit the excited photoconverter that releases energy in the form of non-thermal energy. Efficient PTCA with strong absorption in the near-infrared investigation window (>800 nm) is preferred because near-infrared light can penetrate much deeper into tissues, limiting the interference with blood and tissue, reducing photodamage to normal tissues, and suppressing photon tissue attenuation. An additional measure to enhance the photothermal conversion efficiency is to increase the gather density / structure and light absorption cross section of PTCA at the target tumor site.

[0005] There have been many recent reports on the use of biocompatible photothermal nanomaterials to physically encapsulate organic small molecules such as porphyrins, Ce6 or ICG to improve the pharmacokinetics and tumor targeting efficiency of these small molecule dyes. However, the photothermal conversion efficiency of this class of photothermal materials is lower due to the short excitation wavelength (e.g., 650 nm for porphyrins or Ce6) and therefore less tissue penetration and circulation leakage. Polymer and macromolecular-based PTCAs generally have better photostability than organic small molecule PTCAs or their nanoformulations, especially after the PTCAs self-assemble into nanoparticles (NPs). These resulting nanostructures avoid circulation leakage and rapid clearance from the kidney or liver to some extent. In the past decade, 25-50 nm micellar nanoplatforms have been developed that contain linear PEG and dendritic cholic acid-based amphiphilic polymers (also known as telodendrimers) as building blocks. Several years ago, the development of hybrid telodendrimers containing cholic acid and porphyrins was reported, which can self-assemble to form micellar nanoporphyrins with favorable photothermal properties.

[0006] In general, the application of PTT is limited to the treatment of localized tumors that are accessible to photoirradiation. PTT may also be used intraoperatively to treat the tumor bed when the patient's tumor site is exposed during surgery. Concomitant chemotherapy combined with PTT may potentially be synergistic. With regard to metastatic tumors, PTT may be useful when given in combination with immunotherapy, as the local photothermal effect at the tumor site may activate a systemic antitumor immune response. Durable clinical responses have been seen in some patients treated with checkpoint blockade antibodies. It is well known that the immune checkpoint programmed cell death protein 1 (PD-1) guards against autoimmunity through a dual mechanism that promotes apoptosis of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis of regulatory T cells. Immunotherapy aimed at PD-1 checkpoint strategies may be a promising therapeutic approach for distant metastatic tumors. Although promising, PD-1 checkpoint blockade-based immunotherapy has a limited response rate (approximately 20%) and is only effective in patients whose tumors are pre-infiltrated with T cells. Therefore, synergistic therapeutic strategies combining immunotherapy with other efficient therapeutic approaches have become the current trend in cancer treatment. Recent studies have discovered that PTT can not only directly kill tumor cells upon laser irradiation by inducing apoptosis and necrosis, but also generate antitumor immune responses to improve systemic immunotherapy effects by generating tumor-associated agents from the resected tumor cell residues. Such effects have also been observed in preliminary clinical trial studies.

[0007] Herein, a novel telodendrimer-based photothermal nanoparticle as a high-performance PTCA and controlled delivery system for cancer therapy is reported. Orthotopic OSC-3 oral cancer xenografts and metastatic 4T1 syngeneic breast cancer models were studied to evaluate the synergistic effects of PTT / chemotherapy and PTT / immunotherapy combinations, respectively (Figure 1). The photothermal nanoparticles are a novel type of hybrid telodendrimer (PEG) containing a linear PEG block (Mr ≈ 5000), four dendritic hydrophobic photothermal converters (indocyanine green derivatives, ICGD) and four dendritic cholic acids (CAs) as facial amphiphiles. 5K To further enhance the stability and thermal conversion efficiency of the nanoparticles, another telodendrimer (PEG-CA4-ICGD4, PCI) was prepared, which contains PEG, four cysteines, eight hydrophilic linkers, and eight cholic acids. 5K A CPCI nanoplatinum (PCLC) was added to PCI in a 1:1 ratio and assembled together under a neutral environment to form micelles. The final disulfide-crosslinked micellar nanosystem (CPCI-NP) (Figure 2a) was equipped with ideal smaller size, longer in vivo circulation time, preferential tumor targeting, and deeper tumor penetration. To enhance tumor response, doxorubicin was incorporated into the nanosystem and photothermal / chemotherapeutic combinations were evaluated against orthotopic OSC-3 oral cancer xenograft models. Furthermore, the application of the CPCI nanoplatform loaded with the potent immune agonist imiquimod in combination with PD-1 checkpoint antibody as a potent photoimmunotherapy against 4T1 syngeneic breast cancer model was explored. Summary of the Invention

[0008] Brief Summary of the Invention In one embodiment, the present invention provides a compound of formula (I): [ka] where PEG is a polyethylene glycol (PEG) polymer, each PEG polymer having a molecular weight of 1-100 kDa, each X is a branched monomer unit, and each R 1is independently cholic acid or a cholic acid derivative, and each R 2 and are independently a cyanine.

[0009] In another embodiment, the present invention provides a nanoparticle comprising a plurality of first conjugates, wherein each first conjugate is independently a compound of formula (I) as described herein, and the plurality of conjugates self-assemble in an aqueous medium to form a nanoparticle, such that a hydrophobic core is formed in the interior of the nanoparticle and the PEG of each conjugate self-assembles on the exterior of the nanoparticle.

[0010] In another embodiment, the present invention provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanoparticle of the present invention.

[0011] In another embodiment, the present invention provides a method of treating a disease via photothermal therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a nanoparticle of the present invention and exposing said subject to radiation, thereby treating the disease via photothermal therapy.

[0012] In another embodiment, the present invention provides a method of treating a subject suffering from diseased tissue, the method comprising administering to the subject an effective amount of nanoparticles of the present invention such that the nanoparticles concentrate in the diseased tissue, irradiating the diseased tissue with a first wavelength to identify the diseased tissue, and removing the diseased tissue from the subject, thereby treating the subject.

[0013] In another embodiment, the present invention provides a method of imaging, comprising administering to a subject in need thereof an effective amount of a nanoparticle of the present invention and imaging the subject.

[0014] In another embodiment, the present invention provides a method of preparing a nanoparticle of the present invention comprising forming a reaction mixture comprising a plurality of first conjugates of formula (I) as described herein, wherein the conjugates are allowed to self-assemble to form a nanoparticle such that a hydrophobic core is formed in the interior of the nanoparticle, and the PEG of each conjugate self-assembles on the exterior of the nanoparticle. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows a schematic diagram of combining highly effective photothermal therapeutic nanomedicines (CPIC NPs) with chemotherapy for in situ oral cancer and with immunotherapy for metastatic breast cancer. [Diagram 2] FIG. 2 shows the design and characterization of PCINP and CPIN NP. (a) Schematic of the self-assembly of PCINP and CPCINP; (b) UV-vis-NIR absorption spectra of ICGD molecule and PCI telodendrimer in methanol; DLS and TEM imaging of (c) PCI NPs and (d) CPCI NPs in PBS solution (stained with phosphotungstic acid, telodendrimer concentration: 1 mg mL-1); (e) Continuous dynamic light scattering measurements of PCINP and CPCINP in SDS for 120 min followed by addition of GSH (10 mM); (f) Comparison of the photothermal conversion behaviors of CPCINP, PCI NP and GNR under laser irradiation at 808 nm for 300 s (ICGD concentration: 30 μg mL-1, GNR concentration: 18 μg mL-1); (g) Anti-photobleaching properties of CPCI NP, PCI NP and ICG in PBS solution during five heating-cooling processes (ICGD and ICG concentration: 30 μg mL-1). [Diagram 3]Figure 3 shows the mechanism analysis of CPIN NPs with high photothermal conversion efficiency. Near-infrared fluorescence imaging of (a) PCI NPs and (b) CPCI NPs solutions (10 μL) in the absence of SDS or GSH+SDS and in the presence of different concentrations of SDS or GSH+SDS using an excitation bandpass filter of 735 / 20 nm. Thermal images and quantitative temperature change curves of PCI NPs (c)&(e) and CPCI NPs (d)&(f). Results are expressed as mean ± sd (n=3). After irradiation with NIR laser (808 nm) at 0.8 W cm-2 for 20 s, the temperature was monitored by a thermal camera. (g) Similar equipment and (h) temperature change of CPCINPs and PCLC / TBAI&ICGNPs under laser irradiation of 808 nm (0.8 W cm-2 for 30 s) to investigate the penetration depth and thermal effect. [Figure 4]Figure 4 shows the characterization of CPCI / DOX NPs in vitro and the synergistic cytotoxicity of CPCI / DOX NPs + 808 nm laser against OSC-3 oral cancer cells. (a) Schematic diagram of the self-assembly of CPCI / DOXNPs and DOX release from CPCI / DOXNPs; (b) serum stability of CPCI / DOX NPs in PBS solution at pH 7.4 with / without 10% FBS measured by dynamic light scattering (incubation temperature: 37 °C; data are mean ± sd, n = 3); (c) change in DOX fluorescence intensity and (d) quantitative in vitro DOX release of CPCI / DOXNP aqueous solutions with PBS, GSH (10 mM) and 808 nm laser (0.8 W cm-2, 2 min, 3 times) + GSH (10 mM) (DOX concentration: 0.05 mg mL-1; data are mean ± sd of three parallel samples); (e) uptake of CPCI NPs (green) loaded with DOX (red) in OSC-3 oral cancer cell line. Cells were pretreated with Hoechst 33342 (blue) for 30 min to stain the nuclei and then incubated with CPCI / DOXNPs in PBS. Images were acquired after 6 h using a Deltavision deconvolution microscope (scale bar = 5 μm). (f) Viability (n = 3) of OSC-3 oral cells incubated with CPCI NPs with or without NIR laser irradiation (0.8 W cm-2, 2 min), (g) cell viability and (h) live / dead staining with DiO / PI of OSC-3 oral cells after different treatments: CPCI NPs, 808 nm laser irradiation, free DOX, CPCI / DOX NPs, CPCINPs with 808 nm laser irradiation, CPCI / DOXNPs with 808 nm laser irradiation at a CPCINP concentration of 50 μg mL-1, scale bar = 25 μm, (i) synergistic therapeutic effect and (j) combination index between DOX and CPCINPs + laser for OSC-3 oral cells treatment. [Diagram 5]Figure 5 shows CPCI / DOX NPs tumor images in vivo. (a) In vivo blood clearance kinetics of CPCI NPs, PCLC / TBAI&ICG NPs and free ICG at a dose of 5 mg kg-1 body weight (n=3 in each group). Results are expressed as mean ± sd. (b) ICGD fluorescence images showing tumor accumulation at different time points after tail vein injection and (c) representative in vivo images of nude mice bearing intraepithelial OSC-3 oral cancer labeled by GFP after intravenous injection of CPCI / DOX NPs (DOX dose: 2.5 mg kg-1; total telodendrimer dose: 50 mg kg-1; ICGD dose: 7 mg kg-1). Red arrows point to the tumor site. (d) Representative ex vivo ICGD fluorescence images of organs and tumors excised 48 hours after injection from the same animals, (e) images of distribution of CPCI / DOXNPs in OSC-3 tumor tissues 48 hours after injection observed by laser scanning confocal microscopy. Red: DOX; Green: ICGD, (f) Fluorescence signal intensity of ICGD in organs and tumors at 48 h after injection of CPCI / DOXNPs, (g) DOX distribution in organs and tumor tissues of intraepithelial OSC-3 oral cancer at 24 h, 48 h and 5 days after injection of CPCI / DOXNPs measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Techologies, 7500ce). Three mice were used in each experiment at each time point. Mice received CPCI / DOXNPs at a dose of DOX 2.5 mg kg-1. Results were expressed as mean ± sd. [Figure 6]Figure 6 shows the synergistic antitumor activity of PTT / chemotherapy in mice bearing intraepithelial OSC-3 oral cancer: (a) IR thermal images and (b) average tumor temperature of OSC-3-bearing mice under 808 nm laser irradiation (0.8 Wcm-2) 24 hours after systemic administration of PBS, PCI NPs, CPCI NPs or CPCI / DOX NPs (n=3), (c) schematic diagram of CPCI / DOXNPs-based PTT and chemotherapy to inhibit intraepithelial oral tumor growth, (d) tumor growth curves of intraepithelial OSC-3 oral tumor-bearing mice (5 mice per group), (e) representative photographs of tumor volume changes in the group treated with CPCI / DOXNPs+808 nm laser, (f) histological H&E staining of tumor slices at 28 days after different treatments (scale bar=100 μm×100 and scale bar=25 μm×400). Dose of DOX: 2.5 mg kg-1 and ICGD: 7 mg kg-1. *P values ​​in d were calculated by Tukey's post-hoc test (*P<0.05) by comparing other groups with the last group (CPCI / DOX NPs + 808 nm laser). Data are expressed as mean ± sd. [Figure 7-1] Figure 7 shows the synergistic antitumor activity of photothermal / immunotherapy in mice bearing intraepithelial 4T1 breast cancer (bilateral). (a) Schematic of the proposed mechanism of antitumor immune response induced by CPCI / Imiquimod NPs in combination with α-PD-1 therapy. (b) Encapsulation efficiency of imiquimod into CPCINPs and size change of CPCI / Imiquimod NPs versus drug loading level. The encapsulation efficiency is defined as the ratio of drug loaded into nanoparticles to the initial drug content. The volume of the final solution was kept at 1 mL, and the final concentration of telodendrimers was 20 mg mL-1. (c) Schematic of the synergistic therapeutic effect of photothermal / immunotherapy to inhibit tumor growth at primary and distant sites. [Figure 7-2](d) Primary tumor and (e) distant tumor growth curves of different groups of mice (6 mice per group) with subcutaneous inoculation of 4T1 tumors (imquimod dose: 1.25 mg kg-1, total telodendrimers: 50 mg kg-1 and ICGD: 7 mg kg-1, intraperitoneal injection of 200 μg anti-PD-1 per mouse on days 2 and 8). *P<0.05 in d by comparing group 5 (CPCI / Imiquimod NP + α-PD-1) with group 7 (CPCI / Imiquimod NP + 808 nm laser + α-PD-1), *P<0.05 in e by comparing group 5 (CPCI / Imiquimod NP + α-PD-1) with group 7 (CPCI / NP + 808 nm laser + α-PD-1) and group 4 (CPCI / Imiquimod NP) with group 3 (CPCI / Imiquimod NP + 808 nm laser). (f) Representative photographs of tumor volume changes in mice treated with CPCI / Imiquimod NP + 808 nm laser + α-PD-1. Red and blue circles indicate primary and distant tumors, respectively. [Figure 7-3] (g) H&E stained tumor slices recovered from mice after various treatments as indicated (scale bar = 25 μm). (h) Expression of CD11b, IFN-γ, TNF-α, IL-4, IL-6 and IL-12 in bilateral tumor tissues with different treatments assessed by RT-qPCR (mean ± sd; n = 3). (i) IHC staining of CD8+, PD-1, CD3+ and CD4+ T cells in bilateral tumor tissues recovered with different treatments. Scale bar is 25 μm. [Figure 8] Figure 8 shows the immune memory effect of synergistic photothermal / immunotherapy. (a) Schematic of immune memory by inhibiting cancer recurrence at primary and distant sites, (b) primary tumor and (c) distant tumor growth curves of rechallenge tumors inoculated 50 days after removal of the initial tumor (intraperitoneal injection of 200 μg α-PD-1 per mouse on days 67 and 73). [Figure 9] Figure 9 shows the synthetic approach to ICGD via multi-step chemical reactions. [Figure 10]Figure 10 shows the characterization of the ICGD molecule: (a) chemical structure, (b) 1H NMR spectrum in CDCl3, and (c) mass spectrum of the ICGD molecule measured by ESI mass spectrometry. [Figure 11] Figure 11 shows the characterization of PCI telodendrimers: (a) chemical structure, (b) schematic diagram, and (c) mass spectrum of PCI telodendrimers measured by MALDI-TOF mass spectrometry. [Figure 12] Figure 12 shows the characterization of PCLC telodendrimers: (a) chemical structure, (b) schematic diagram, and (c) mass spectrum of PCLC telodendrimers measured by MALDI-TOF mass spectrometry. [Figure 13] Figure 13 shows (a) fluorescence spectra of ICGD and PCI telodendrimer in methanol, (b) UV-vis-NIR absorption spectra of ICGD and PCI telodendrimer in methanol and PCI NPs in PBS (ICGD concentration: 30 μg mL-1), (c) fluorescence spectra of ICGD from PCI NPs in PBS and SDS (PCI telodendrimer concentration: 0.1 mg mL-1), (d) fluorescence spectra of ICGD from CPCI NPs in PBS, SDS, GSH and GSH+SDS (PCI telodendrimer concentration: 0.1 mg mL-1). [Figure 14] Figure 14 shows (a) the encapsulation efficiency of DOX into CPCI NPs and the size change of CPCI / DOX NPs versus drug loading level. The encapsulation efficiency is defined as the ratio of drug loaded into the nanoparticles to the initial drug content. The final solution volume was kept at 1 mL, and the final concentration of telodendrimers was 20 mg / mL. (b) Schematic diagram and representative photographs of CPCINPs and CPCI / DOX NPs in PBS solution (total telodendrimers concentration: 20 mg mL-1; DOX concentration: 1 mg mL-1). [Figure 15] FIG. 15 shows blood test parameters for white blood cells (WBC), lymphocytes, eosinophils, red blood cell distribution width (RDW), platelets and mean platelet volume (MPV) at 24 and 72 hours in healthy Balb / c mice intravenously injected with three different concentrations of CPCINP. [Figure 16] FIG. 16 shows blood test parameters for liver function alanine aminotransferase, albumin, alkaline phosphatase, aspartate transaminase, blood urea nitrogen, creatinine, bilirubin, total protein, cholesterol, high density lipoprotein, low density lipoprotein and triglycerides in healthy Balb / c mice intravenously injected with three different concentrations of CPCINP for 24 and 72 hours. [Figure 17] Figure 17 shows the detection of apoptotic OSC-3 buccal cells using Annexin V-FITC and PI flow cytometry assays in various cell culture conditions. Q1: necrotic cells, Q2: late apoptotic cells, Q3: normal live cells, Q4: early apoptotic cells. DOX dose: 0.005 mgmL-1; 808 nm laser irradiation: 0.8 Wcm-2, 2 min. [Figure 18] Figure 18 shows the weight curves of experimental mice bearing intraepithelial OSC-3 oral cancer receiving different therapeutic formulations via the tail vein at 3-day intervals (n=5; DOX dose: 2.5 mg kg and ICGD dose: 7 mg kg; data were the mean ± standard error of three parallel samples). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention I. Definition Unless otherwise specified, 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. Furthermore, any methods or materials similar or equivalent to those described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

[0017] As used herein, "A," "an," or "the" not only includes embodiments having one element, but also includes embodiments having a plurality of elements. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to an "agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0018] As used herein, the terms "dendrimer" and "dendritic polymer" refer to a branched polymer comprising a focal point, a plurality of branched monomer units, and a plurality of end groups. The monomers are bonded to each other to form arms (or "dendrons") extending from the focal point and terminating in end groups. The dendrimer focal point can be bonded to other segments of the compounds of the invention, and the end groups can be further functionalized with additional chemical moieties.

[0019] As used herein, the term "telodendrimer" refers to a dendrimer comprising a hydrophilic PEG segment and one or more chemical moieties covalently attached to one or more terminal groups of the dendrimer. These moieties include, but are not limited to, hydrophobic groups, hydrophilic groups, amphiphilic compounds, and organic moieties. An orthogonal protecting group strategy can be used to selectively place different moieties at the desired terminal groups.

[0020] As used herein, the terms "monomer", "monomer unit" or "branched monomer unit" refer to diamino carboxylic acid, dihydroxy carboxylic acid or hydroxyl amino carboxylic acid. Examples of diamino carboxylic acid groups of the present invention include, but are not limited to, 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and 5-amino-2-(3-aminopropyl)pentanoic acid. Examples of dihydroxy carboxylic acid groups of the present invention include, but are not limited to, glyceric acid, 2,4-dihydroxybutyric acid, glyceric acid, 2,4-dihydroxybutyric acid, 2,2-bis(hydroxymethyl)propionic acid and 2,2-bis(hydroxymethyl)butyric acid. Examples of hydroxylamino carboxylic acids include, but are not limited to, serine and homoserine. One of skill in the art will appreciate that other monomer units are useful in the present invention.

[0021] As used herein, the term "amino acid" refers to a carboxylic acid having an amine functional group. Amino acids include the diamino carboxylic acids described above. Amino acids include naturally occurring α-amino acids, in which the amine is attached to the carbon adjacent to the carbonyl carbon of the carboxylic acid. Examples of naturally occurring α-amino acids include, but are not limited to, L-aspartic acid, L-glutamic acid, L-histidine, L-lysine, and L-arginine. Amino acids can also include the D-enantiomers of naturally occurring α-amino acids, as well as β-amino acids and other non-naturally occurring amino acids.

[0022] As used herein, the term "cholic acid" refers to (R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydrop-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid. Cholic acid is also known as 3α,7α,12α-trihydroxy-5β-cholanic acid, 3-α,7-α,12-α-trihydroxy-5-cholan-24-oic acid, 17-β-(1-methyl-3-carboxypropyl)etiocholan-3α,7α,12α-triol, cholalic acid, and coraline. Cholic acid derivatives and analogs such as allocholic acid, pithocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholic acid are also useful in the present invention. Cholic acid derivatives can be designed to modulate the properties of the nanocarriers resulting from the telodendrimer assemblies, such as micelle stability and membrane activity, For example, cholic acid derivatives can have a hydrophilic face modified with one or more glycerol, aminopropanediol, or other groups.

[0023] As used herein, the term "cyanine" refers to a family of synthetic dyes belonging to the polymethine group. Cyanines can be used as fluorescent dyes for biomedical imaging. Cyanines can be streptocyanines (also known as open-chain cyanines), hemicyanines, and closed cyanines. Closed cyanines each have a nitrogen that is independently part of a heteroaromatic moiety.

[0024] As used herein, the term "nanoparticle" or "nanocarrier" refers to a micelle resulting from the aggregation of the dendrimer conjugates of the invention. Nanocarriers have a hydrophobic core and a hydrophilic exterior.

[0025] As used herein, the term "amphiphilic compound" refers to a compound that has both hydrophobic and hydrophilic portions. For example, the amphiphilic compound of the present invention can have one hydrophilic face of the compound and one hydrophobic face of the compound. Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives.

[0026] As used herein, the term "crosslinkable group" or "crosslinking group" refers to a functional group capable of bonding to a similar or complementary group on another molecule, e.g., a first crosslinkable group on a first dendritic polymer linking to a second crosslinkable group on a second dendritic polymer. "Thiol-containing crosslinkable group" refers to a crosslinkable group that includes a sulfur atom. Groups suitable as crosslinkable and crosslinking groups in the present invention when incorporated into the interior of a dendrimer include thiols such as cysteine, boronic acids, and 1,2-diols including 1,2-dihydroxybenzenes such as catechol. Groups suitable as crosslinkable and crosslinking groups in the present invention when incorporated into one or more terminal groups of a dendrimer include thiols such as cysteine ​​and N-acetylcysteine. When the crosslinkable and crosslinking groups are combined, they form crosslinks such as disulfides and boronic esters. Other crosslinkable and crosslinking groups are suitable in the present invention.

[0027] As used herein, the term "linking group" refers to a chemical moiety that links one segment of a dendrimer conjugate to another. The types of bonds used to link the linker to the dendrimer segments include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. One of skill in the art will appreciate that other types of bonds are useful in the present invention.

[0028] As used herein, the term "drug" or "therapeutic agent" refers to an agent that can treat and / or ameliorate a condition or disease. The drug can be a hydrophobic drug, which is any drug that repels water. Hydrophobic drugs useful in the present invention include, but are not limited to, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, Hondoshan A, Hondoshan B, or Hondoshan C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporine 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. The drugs of the present invention also include prodrug forms. Those skilled in the art will understand that other drugs are useful in the present invention.

[0029] As used herein, the terms "treat," "treating," and "treatment" refer to an indication of success in treating or ameliorating an injury, condition, state, or symptom (e.g., pain), including any objective or subjective parameter, such as relief, remission, relief of symptoms, or making the symptom, injury, condition, or the like 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 or condition. The treatment or amelioration of a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0030] As used herein, the term "administering" refers to 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, into a subject.

[0031] As used herein, the term "therapeutically effective amount or dose" or "amount or dose sufficient for treatment" or "effective or sufficient amount or dose" refers to the dose that produces the therapeutic effect administered. The exact dose depends on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vol. 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.

[0032] As used herein, the term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human.

[0033] As used herein, the term "disease" refers to an abnormal condition that is not due to external insult and adversely affects the structure or function of part or all of an organism. A disease is often interpreted as a medical condition associated with certain symptoms and signs. Diseases can include cancer, immune deficiencies, hypersensitivity, allergies, and autoimmune diseases.

[0034] As used herein, the term "diseased tissue" refers to cellular tissue that is part of a disease as described herein.

[0035] As used herein, the term "checkpoint blocking antibodies" refers to antibodies that inhibit immune checkpoints, which are key regulators of the immune system that, when stimulated, can attenuate the immune response to immune stimulation. Checkpoint blocking antibodies can block inhibitory checkpoints and restore immune system function. Examples of checkpoint blocking antibodies include, but are not limited to, anti-PD-1, anti-PDL-1, anti-PDL-2, and anti-CTLA-4.

[0036] As used herein, the term "photothermal therapy" refers to the use of non-toxic, photosensitive compounds that generate heat upon exposure to light. Like photodynamic therapy, photothermal therapy involves a photosensitizer and a light source, typically infrared light. However, photothermal therapy does not require oxygen. A variety of photosensitizers can be used, including porphyrins, chlorophyll, and pigments.

[0037] As used herein, the term "radiation" refers to the emission or transmission of energy in the form of waves or particles through space or a material medium, and "irradiation" refers to the process by which an object is exposed to radiation. In some examples, irradiation includes the use of infrared radiation.

[0038] II. Cyanine telodendrimers In some embodiments, the present invention provides a compound of formula (I): [ka] where PEG is a polyethylene glycol (PEG) polymer, each PEG polymer having a molecular weight of 1-100 kDa, each X is a branched monomer unit, and each R 1 is independently cholic acid or a cholic acid derivative, and each R 2 and are independently a cyanine.

[0039] Polyethylene glycol (PEG) polymers of any size and structure are useful in the telodendrimers of the present invention. In some embodiments, the PEG is 1-100 kDa. In some embodiments, the PEG is 1-50 kDa. In some embodiments, the PEG is 1-10 kDa. In some embodiments, the PEG is about 10 kDa, about 9 kDa, about 8 kDa, about 7 kDa, about 6 kDa, about 5 kDa, about 4 kDa, about 3 kDa, about 2 kDa, or about 1 kDa. In some embodiments, the PEG is about 5 kDa. One of skill in the art will appreciate that other PEG polymers and other hydrophilic polymers are useful in the present invention. The PEG can be of any suitable length.

[0040] Dendritic polymers can be made from branching monomer units X, including amino acids or other bifunctional AB2 type monomers, where A and B are two different functional groups that can react together such that the resulting polymer chain has a branch point where an AB bond is formed. In some embodiments, each branching monomer unit X can be a diamino carboxylic acid, a dihydroxy carboxylic acid, and a hydroxyl amino carboxylic acid. In some embodiments, X is a diamino carboxylic acid. In some embodiments, each diamino carboxylic acid can be 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid, or 5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, each dihydroxycarboxylic acid can be glyceric acid, 2,4-dihydroxybutyric acid, 2,2-bis(hydroxymethyl)propionic acid, 2,2-bis(hydroxymethyl)butyric acid, serine, or threonine. In some embodiments, each hydroxylaminocarboxylic acid can be serine or homoserine. In some embodiments, the diaminocarboxylic acid is an amino acid. In some embodiments, each branching monomer unit X is a diaminocarboxylic acid and each diaminocarboxylic acid is lysine.

[0041] Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives. "Cholic acid" refers to (R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid, having the following structure: [ka]

[0042] Cholic acid derivatives and analogs include, but are not limited to, allocholic acid, pithocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholic acid. Cholic acid derivatives can be designed to modulate the properties of nanocarriers resulting from telodendrimer assemblies, such as micelle stability and membrane activity. For example, cholic acid derivatives can have hydrophilic faces modified with one or more glycerol, aminopropanediol, or other groups.

[0043] In some embodiments, each R 1 is independently cholic acid, (3α,5β,7α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid (CA-4OH), (3α,5β,7α,12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid (CA-5OH), or (3α,5β,7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholic acid (CA-3OH-NH2). 1 is cholic acid.

[0044] The cyanines of the present invention refer to a family of synthetic dyes belonging to a polymethine group containing at least one quaternary amine. The cyanines can be used for imaging. In some embodiments, the cyanines can be streptocyanines, hemicyanines, or closed cyanines. The hemicyanines can have the following structure: [ka] (where n is an integer from 0 to 5, and ring A is a heteroaromatic moiety). The closed cyanines, each having a nitrogen that is independently part of a heteroaromatic moiety, can have the following structure: [ka] where n is an integer from 0 to 5, and ring A is independently a heteroaromatic moiety. The heteroaromatic moiety can be pyrrole, imidazole, thiazole, pyridine, quinolone, indole, benzoindole, or benzothiazole, each of which is optionally selected from the group consisting of C 1-6 Each R group may be substituted with 1 to 4 functional groups selected from the group consisting of alkyl and sulfonate. 1-10 Alkyl (CH2) m SO3Na and (CH2) m COOH, where m is an integer from 1 to 10.

[0045] In some embodiments, each R 2 is independently cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, cyanine 7.5, indocyanine green, or an indocyanine green derivative having the structure (S1). [ka] In some embodiments, each R 2 is a cyanine having the structure (S1).

[0046] In some embodiments, the compound can be a compound of formula (I), where each PEG has a molecular weight of 5 kDa, each X is lysine, and each R 1 is cholic acid, and each R 2 is independently a cyanine comprising the structure (S1). In some embodiments, the compound has the structure: [ka]

[0047] III. Nanoparticles The telodendrimers of the invention provide for aggregation to form nanocarriers comprising a hydrophobic core and a hydrophilic exterior. In some embodiments, the invention provides nanocarriers having an interior and an exterior, the nanocarriers comprising one or more dendrimer conjugates of the invention, each compound self-assembles in an aqueous medium to form a nanocarrier, such that a hydrophobic pocket is formed in the interior of the nanocarrier and the PEG of each compound self-assembles on the exterior of the nanocarrier.

[0048] In some embodiments, the present invention provides a nanoparticle comprising a plurality of first conjugates, where each first conjugate is independently a compound of Formula (I) as described herein, and the plurality of conjugates self-assemble in an aqueous solvent to form a nanoparticle, such that a hydrophobic core is formed in the interior of the nanoparticle and the PEG of each conjugate self-assembles on the exterior of the nanoparticle.

[0049] A plurality of first conjugates useful in the present invention comprises a conjugate of formula (I) as described herein. In some embodiments, the nanoparticle further comprises a plurality of second conjugates, each second conjugate being independently a compound of formula (II). [ka] wherein PEG is a polyethylene glycol (PEG) polymer, each PEG polymer having a molecular weight of 1-100 kDa, each X is a branched monomer unit, each Y is a thiol-containing crosslinkable group, each L is a linking group, and each R 1 are independently cholic acid or a cholic acid derivative).

[0050] Polyethylene glycol (PEG) polymers of any size and structure are useful in the telodendrimers of the present invention for compounds of formula II. In some embodiments, the PEG is 1-100 kDa. In some embodiments, the PEG is 1-50 kDa. In some embodiments, the PEG is 1-10 kDa. In some embodiments, the PEG is about 10 kDa, about 9 kDa, about 8 kDa, about 7 kDa, about 6 kDa, about 5 kDa, about 4 kDa, about 3 kDa, about 2 kDa, or about 1 kDa. In some embodiments, the PEG is about 5 kDa. One of skill in the art will appreciate that other PEG polymers and other hydrophilic polymers are useful in the present invention. The PEG can be of any suitable length.

[0051] Branching monomer units useful in the present invention include any suitable amino acid or other bifunctional AB2 type monomer, where A and B are two different functional groups that can react together so that the resulting polymer chain has a branch point where an AB bond is formed. In some embodiments, each branching monomer unit X can be a diamino carboxylic acid, a dihydroxy carboxylic acid, and a hydroxyl amino carboxylic acid. In some embodiments, X is a diamino carboxylic acid. In some embodiments, each diamino carboxylic acid can be 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid, or 5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, each dihydroxycarboxylic acid can be glyceric acid, 2,4-dihydroxybutyric acid, 2,2-bis(hydroxymethyl)propionic acid, 2,2-bis(hydroxymethyl)butyric acid, serine, or threonine. In some embodiments, each hydroxylaminocarboxylic acid can be serine or homoserine. In some embodiments, the diaminocarboxylic acid is an amino acid. In some embodiments, each branching monomer unit X is a diaminocarboxylic acid and each diaminocarboxylic acid is lysine.

[0052] The linking group L can include any suitable linker. Generally, the linking group is a bifunctional linker, having two functional groups for reacting with each of the two telodendrimer segments. In some examples, the linking group can be a heterobifunctional linker. In some examples, the linking group can be a homobifunctional linker. In some examples, the linking group L can be polyethylene glycol, polyserine, polyglycine, poly(serine-glycine), an aliphatic amino acid, 6-aminohexanoic acid, 5-aminopentanoic acid, 4-aminobutanoic acid, or β-alanine. In some embodiments, the linking group L is an Ebes linker having the formula: [ka]

[0053] One of skill in the art will recognize that the size and chemical nature of the linking group can vary based on the structure of the telodendrimer segments being linked.

[0054] In some embodiments, the nanoparticles of the present invention each R 1 Each R is independently a cholic acid or a cholic acid derivative. 1 is independently cholic acid, (3α,5β,7α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid (CA-4OH), (3α,5β,7α,12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid (CA-5OH), or (3α,5β,7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholic acid (CA-3OH-NH2). 1 is cholic acid.

[0055] In some embodiments, the nanoparticle comprises a first conjugate of Formula (I), wherein each PEG has a molecular weight of 5 kDa, each X is lysine, and each R 1 is cholic acid, and each R2 is a cyanine having the structure (S1). In some embodiments, the nanoparticle further comprises a second conjugate of formula (II), wherein each PEG has a molecular weight of 5 kDa, each X is a lysine, each Y is a cysteine, and each L is a cyanine having the formula: [ka] and each R 1 is cholic acid.

[0056] In some embodiments, the nanoparticles of the present invention are crosslinked via a thiol-containing crosslinkable group of formula (II). In some examples, the thiol-containing crosslinkable group is cysteine ​​or N-acetyl-cysteine. In some examples, the thiol-containing crosslinkable group is cysteine.

[0057] In some examples, the nanoparticles of the present invention comprise conjugates of formula (I) and formula (II) in a ratio (w / w) of about 100:1 to 1:100, about 50:1 to 1:50, about 25:1 to 1:25, about 10:1 to about 1:10, or about 1:1. In some examples, the conjugate of formula (I) to the conjugate of formula (II) has a ratio (w / w) of about 100:1, 50:1, 25:1, 10:1, or 1:1. In some embodiments, the conjugates of formula (I) and formula (II) are in a ratio of about 1:1 (w / w).

[0058] In some instances, the average diameter of the nanoparticles is about 250 nm, about 100 nm, about 50 nm, or about 20 nm, hi some embodiments, the average diameter of the nanoparticles is about 20 nm.

[0059] In some embodiments, the nanoparticles of the present invention further comprise a therapeutic agent within the hydrophobic core. The therapeutic agent can be poorly water soluble. In some examples, the therapeutic agent is selected from the group consisting of bortezomib, paclitaxel, SN38, camptothecin, etoposide and doxorubicin, docetaxel, VP16, prednisone, dexamethasone, vincristine, vinblastine, temsirolimus, carmusine, gardiquimod, doxorubicin, daunorubicin, vincristine, vinblastine, paclitaxel, SN-38, imiquimod, resiquimod, motolimod, lenalidomide, pomalidomide, and LLS30 having the structure (S2). [ka]

[0060] In some embodiments, the therapeutic agent is selected from the group consisting of gardiquimod, doxorubicin, daunorubicin, vincristine, vinblastine, paclitaxel, SN-38, imiquimod, resiquimod, motolimod, lenalidomide, pomalidomide, and LLS30 having the structure (S2).

[0061] IV.Treatment The nanoparticles of the present invention can be used to treat disease. The nanoparticles of the present invention can be used to treat disease via photothermal therapy. The nanoparticles of the present invention can also be used to treat subjects suffering from diseased tissue. The nanoparticles of the present invention can also be used for imaging by sequestering contrast agents inside the nanocarrier or by binding contrast agents to the conjugates of the nanocarrier.

[0062] In some embodiments, the present invention provides a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nanoparticle of the present invention.

[0063] The nanoparticles of the invention can be administered to a subject for the treatment of diseases including cancer, including, but not limited to, carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, 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's lymphoma, and non-Hodgkin's lymphoma (see CANCER: PRINCIPLES AND METHODS FOR ADDITIONAL CANCER). PRACTICE (DeVita, VT et al., 2008).

[0064] Other diseases that can be treated by the nanocarriers of the present invention include: (1) inflammatory or allergic diseases such as systemic anaphylaxis or hypersensitivity reactions, drug allergies, insect sting allergies, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis and enteritis, vaginitis, psoriasis and dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, vasculitis, spondyloarthropathy, scleroderma, asthma, allergic rhinitis, hypersensitivity pulmonary disease, and other respiratory allergic diseases; (2) arthritis (rheumatic and psoriatic) (3) autoimmune diseases such as osteoarthritis, multiple sclerosis, systemic lupus erythematosus, diabetes mellitus, glomerulonephritis, (4) other diseases in which undesirable inflammatory responses should be inhibited (e.g., atherosclerosis, myositis, neurological conditions such as stroke and closed head injury, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis, and Behcet's syndrome).

[0065] In some embodiments, the invention provides a method of treating a disease, wherein the disease is cancer, hi some embodiments, the disease is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma, or metastatic cancer.

[0066] In some embodiments, the present invention provides a method of treating a disease further comprising a therapeutic agent within the hydrophobic core. Therapeutic agents suitable for the methods of the present invention are described herein above. In some embodiments, the method of treating a disease comprises a therapeutic agent selected from the group consisting of doxorubicin, gardiquimod, and imiquimod. In some embodiments, the method of treating a disease comprises a therapeutic agent, the therapeutic agent being imiquimod, and further comprises administering a checkpoint blockade antibody.

[0067] Checkpoint blockade antibodies useful in the present invention include, but are not limited to, anti-PD-1, anti-PDL1, anti-CLTA-4, nivolumab, pembrolizumab, Keytruda, spartalizumab, atezolizumab, and ipilimumab. In some embodiments, the checkpoint blockade antibody useful in the present invention is anti-PD-1, anti-PDL1, or anti-CTLA-4. In some embodiments, the checkpoint blockade antibody is anti-PD-1.

[0068] In some embodiments, the present invention provides a method of treating a disease via photothermal therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a nanoparticle of the present invention and exposing the subject to radiation, thereby treating the disease via photothermal therapy.

[0069] The method of treatment using the nanocarriers of the present invention also includes treating a disease by photothermal therapy. This method generally includes administering the nanocarriers of the present invention to a subject and then exposing the subject to radiation of a specific wavelength to induce photodynamic therapy or photothermal therapy depending on the wavelength of the light. Upon exposure to radiation or light, the nanoparticles generate heat sufficient for photothermal therapy. In some embodiments, the present invention provides a method of treating a disease via photothermal therapy, the method includes administering a therapeutically effective amount of the nanocarriers of the present invention to a subject in need thereof and exposing the subject to radiation, thereby treating the disease via photothermal therapy. In some embodiments, the method is a method of treating a disease via photothermal therapy.

[0070] Radiation useful in the methods of the present invention includes electromagnetic radiation, particle radiation, acoustic radiation, and gravitational radiation. Examples of electromagnetic radiation include, but are not limited to, radio waves, microwaves, infrared light, visible light, ultraviolet light, X-rays, and gamma rays.

[0071] In some embodiments, the present invention provides methods of treating disease via photothermal therapy, where a subject is exposed to electromagnetic radiation, hi some embodiments, the present invention provides methods of treating disease via photothermal therapy, where a subject is exposed to near infrared light irradiation.

[0072] The nanoparticles of the present invention can be administered to a subject to treat a disease via photothermal therapy, the disease being as described herein above.

[0073] In some embodiments, the present invention provides a method of treating a disease via photothermal therapy, wherein the disease is cancer, hi some embodiments, the disease is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma, or metastatic cancer.

[0074] In some embodiments, the present invention provides a method of treating a disease via photothermal therapy, further comprising a therapeutic agent in the hydrophobic core. Therapeutic agents suitable for the methods of the present invention are described herein above. In some embodiments, the method of treating a disease comprises a therapeutic agent selected from the group consisting of doxorubicin, gardiquimod, and imiquimod. In some embodiments, the method of treating a disease comprises a therapeutic agent, and the therapeutic agent is imiquimod, and further comprises administering an anti-PD-1 via injection.

[0075] In some embodiments, the present invention provides a method of treating a subject suffering from diseased tissue, the method comprising administering to the subject an effective amount of nanoparticles of the present invention such that the nanoparticles concentrate in the diseased tissue, irradiating the diseased tissue with a first wavelength to identify the diseased tissue, and removing the diseased tissue from the subject, thereby treating the subject.

[0076] In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, the method further comprising delaying the irradiating step at least 1 hour after the administering step, at least 2 hours after the administering step, at least 3 hours after the administering step, at least 4 hours after the irradiating step, at least 5 hours after the irradiating step, or at least 6 hours after the irradiating step. In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, the method further comprising delaying the irradiating step at least 3 hours after the administering step.

[0077] In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, the method further comprising irradiating any remaining diseased tissue in the subject with a second wavelength, hi some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, where the removal step and both irradiating steps are performed during a single procedure on the subject.

[0078] In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, wherein the first wavelength and the second wavelength are each independently in the ultraviolet spectrum, the visible spectrum, or the infrared spectrum. In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, wherein the first wavelength and the second wavelength are each independently in the extreme ultraviolet spectrum, the near ultraviolet spectrum, the near infrared spectrum, the mid infrared spectrum, or the far infrared spectrum. In some embodiments, the invention provides a method of treating a subject suffering from diseased tissue, wherein the first wavelength and the second wavelength are each independently in the near infrared spectrum.

[0079] In some embodiments, the present invention provides a method of treating a subject suffering from diseased tissue, where the diseased tissue is any suitable diseased tissue from a disease described herein. In some embodiments, the present invention provides a method of treating a subject suffering from diseased tissue, where the diseased tissue is cancer. In some embodiments, the diseased tissue is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma, or metastatic cancer. In some embodiments, the present invention provides a method of treating a subject suffering from diseased tissue, where the diseased tissue is ovarian cancer.

[0080] In some embodiments, the present invention provides a method of imaging, the method comprising administering to a subject in need thereof an effective amount of a nanoparticle of the present invention and imaging the subject. In some embodiments, the nanoparticle can further comprise an imaging agent within the hydrophobic core.

[0081] Exemplary contrast agents include paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are contrast agents that become magnetic under an externally applied magnetic field. Examples of paramagnetic agents include, but are not limited to, iron particles, including nanoparticles. Optical probes are fluorescent compounds that can be detected by excitation with radiation of one wavelength and detection with radiation of a second, different wavelength. Optical probes useful in the present invention include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo radioactive decay. Radionuclides useful in the present invention include, but are not limited to, 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 is one example.

[0082] V. Nanoparticle Preparation Method In some embodiments, the present invention provides a method of preparing a nanoparticle of the present invention comprising forming a reaction mixture comprising a plurality of first conjugates of formula (I) as described herein, wherein the conjugates form a nanoparticle such that a hydrophobic core is formed in the interior of the nanoparticle and the PEG of each conjugate self-assembles on the exterior of the nanoparticle.

[0083] In some embodiments, the present invention provides a method of preparing a nanoparticle of the present invention, further comprising a plurality of second conjugates of formula (II) as described herein.

[0084] In some embodiments, the present invention provides a method for preparing the nanoparticles of the present invention, wherein the aqueous solution is a buffer solution.The buffer solution can include a combination of buffering agents, including but not limited to citrate, acetic acid, KH2PO4, CHES, borate, TAPS, bicine, tris, tricine, TAPS, HEPES, TES, MOPS, PIPES, cacodylate, and MES.In some embodiments, the aqueous solution includes PBS.

[0085] In some embodiments, the present invention provides a method of preparing the nanoparticles of the present invention, the method further comprising sonicating the reaction mixture. The reaction mixture can be sonicated for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, or about 1 hour. In some embodiments, the reaction mixture can be sonicated for about 10 minutes.

[0086] In some embodiments, the present invention provides a method of preparing the nanoparticles of the present invention, wherein the aqueous solution is PBS, and the method further comprises sonicating the reaction mixture for about 10 minutes.

[0087] In some examples, the present invention provides a method of preparing a nanoparticle of the present invention, wherein the nanoparticle comprises a conjugate of Formula (I) and Formula (II) in a ratio (w / w) of about 100:1 to 1:100, about 50:1 to 1:50, about 25:1 to 1:25, about 10:1 to about 1:10, or about 1:1. In some examples, the conjugate of Formula (I) / Formula (II) has a ratio (w / w) of about 100:1, 50:1, 25:1, 10:1, or 1:1. In some embodiments, the conjugate of Formula (I) and Formula (II) is in a ratio of about 1:1 (w / w).

[0088] In some embodiments, the present invention provides a method of preparing a nanoparticle, wherein the nanoparticle further comprises a plurality of second conjugates of formula (II), and the thiol-containing crosslinkable group is cysteine ​​or N-acetylcysteine. In some examples, the thiol-containing crosslinkable group is cysteine. In some embodiments, the crosslink is formed via oxidation of cysteine.

[0089] VI. Formulations and Pharmaceutical Compositions The compositions of the present invention can be prepared in a wide variety of oral, parenteral and topical dosage forms. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, troches, 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 intranasally, for example, by inhalation. Additionally, the compositions of the present invention can be administered transdermally. The compositions of the present invention can also be administered by intraocular, intravaginal and intrarectal routes, including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Thus, the present invention also provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient and a composition of the present invention.

[0090] For preparing pharmaceutical compositions from the compounds of the present invention, pharma- ceutical acceptable carriers can be either solid or liquid. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A 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 of formulation and administration techniques are fully described in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0091] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted into the desired shape and size. Powders and tablets preferably contain 5% or 10% to 70% of the composition of the present invention.

[0092] Suitable solid excipients include, but are not limited to, magnesium carbonate, magnesium stearate, talc, pectin, dextrin, starch, tragacanth, low melting waxes, cocoa butter, carbohydrates, sugars (including, but not limited to, lactose, sucrose, mannitol or sorbitol), starches from corn, wheat, rice, potato or other plants, celluloses such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose, gums including gum arabic and tragacanth, and proteins including, but not limited to, gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.

[0093] Dragee cores are provided with suitable coatings such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions that may contain suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the amount of active compound (i.e., dosage). The pharmaceutical formulations of the present invention can also be used orally using push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and coatings such as glycerol or sorbitol. Push-fit capsules can contain the compound of the present 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 compositions of the present invention can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0094] For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the composition of the present invention is dispersed homogeneously therein, as by stirring, etc. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

[0095] 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.

[0096] Aqueous solutions suitable for oral use can be prepared by dissolving the composition of the present invention in water and adding suitable colorants, flavorings, stabilizers and thickeners as required. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan 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 preparation may be adjusted for osmotic pressure.

[0097] Also included are solid form preparations that are intended to be converted immediately before use into liquid form preparations for oral administration.Such liquid forms include solutions, suspensions and emulsions.These preparations can contain, in addition to the active ingredient, colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0098] Oil suspensions can be formulated by suspending the compounds of the present invention in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils, such as liquid paraffin, or mixtures thereof. Oil suspensions can contain thickening agents, such as beeswax, paraffin, or cetyl alcohol. Sweeteners, such as glycerol, sorbitol, or sucrose, can be added to provide a palatable oral preparation. These preparations can be preserved by the addition of an antioxidant, such as ascorbic acid. For examples of injectable oil vehicles, 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 naturally occurring gums such as gum acacia and gum tragacanth, naturally occurring 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.

[0099] The composition of the present invention can also be delivered as microspheres for sustained release in the body.For example, microspheres can be formulated to be administered via intradermal injection of drug-containing microspheres that slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995), as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995), or as oral microspheres (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).Both transdermal and intradermal routes provide constant delivery for weeks or months.

[0100] In some embodiments, the compositions of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a cavity of a body cavity or organ. Formulations for administration will generally comprise a solution of the compositions of the present invention dissolved in a pharma- ceutically acceptable carrier. Acceptable vehicles and solvents that can be used include water and Ringer's solution, isotonic sodium chloride. Additionally, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid can be used in the preparation of injectables as well. These solutions are sterile and generally free of undesirable matter. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations can include pharma-ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the compositions of the present invention in these formulations can vary widely and is selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular mode of administration selected and the needs of the patient. For IV administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension.This suspension can be formulated according to known techniques using their appropriate dispersing or wetting agent and suspending agent.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.

[0101] In some embodiments, the formulation of the composition of the present invention can be delivered by using liposomes that fuse with cell membranes or are endocytosed, i.e., by using ligands attached to the liposomes or directly attached to the oligonucleotides that bind to cell surface membrane protein receptors and cause endocytosis.The composition of the present invention can be delivered to target cells in vivo by using liposomes, especially when the liposome surface has a ligand specific to the target cells or is otherwise preferentially directed to a particular organ (see, for example, Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

[0102] VII. Administration The compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical. Topical transdermal administration methods can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols.

[0103] The pharmaceutical preparation is preferably in unit dosage form.In such dosage form, the preparation is subdivided into unit doses containing appropriate amounts of the composition of the present invention.The unit dosage form can be a packaged preparation, the package containing discrete amounts of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules.Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or the appropriate number of any of these packaged forms.

[0104] The compounds of the present invention can be present in any suitable amount and can depend on a variety of factors, including, but not limited to, the weight and age of the subject, the disease state, etc. Suitable dosage ranges of the compounds of the present 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 dosages of the compounds of the present 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.

[0105] The compounds of the invention can be administered at any suitable frequency, interval and duration. For example, the compounds of the invention can be administered once per hour, or 2, 3 or more times per hour, once per day, or 2, 3 or more times per day, or once per 2, 3, 4, 5, 6 or 7 days, thereby providing 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, and 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours. The compounds of the invention can be administered once, twice or three or more times for 1 hour, 1-6 hours, 1-12 hours, 1-24 hours, 6-12 hours, 12-24 hours, 1 day, 1-7 days, 1 week, 1-4 weeks, 1 month, 1-12 months, 1 year or more, or even indefinitely.

[0106] The compositions may also include other compatible therapeutic agents. The compounds described herein may be used in combination with each other, with other active agents, or with adjuvants that may not be effective alone but may contribute to the effectiveness of the active agent.

[0107] The compound of the present invention can be co-administered with another active agent. Co-administration includes administering 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. Co-administration also includes administering the compound of the present invention and the active agent simultaneously, about simultaneously (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 dosage level per day.

[0108] In some embodiments, coadministration can be achieved by co-formulation, i.e., preparing a single pharmaceutical composition that includes both the compound of the invention and the active agent, hi some embodiments, the compound of the invention and the active agent can be formulated separately.

[0109] The compounds of the invention and the active agent can be present 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 the compositions of the invention 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 dosage amounts and dosage ratios of the compounds of the invention and the other active agents are suitable in the compositions and methods of the invention. EXAMPLES

[0110] VIII. Examples result Design and preparation of CPCI-NPs To impart photothermal properties, indocyanine green derivatives (ICGD), a highly stable hydrophobic photothermal conversion agent, were first synthesized and introduced into the telodendrimer system (Figure 9). As shown in Figures 10a, 10b, and 10c, 1The chemical structure of ICGD was verified by H NMR and ESI mass spectrometry measurements. From the UV-vis absorption spectrum, ICGD showed a broad absorption band from 500 to 900 nm in methanol with two absorption peaks. One shoulder peak was at about 720 nm, and the other peak was in the near-infrared range at about 785 nm (Figure 2b). A fluorescence emission spectrum with a peak value of 835 nm was obtained in methanol upon excitation at 808 nm. The ICGD molecule was functionalized with a carboxylic acid group, whereby four ICGD units were attached to PEG. 5k PEG can be easily grafted onto the -CA4-telodendrimers. 5K The -CA4-ICGD4(PCI) telodendrimer was obtained, and its molecular weight was determined to be 10,430 Da by MALDI-TOF mass spectrometry (Figures 11a and 11b). The absorption and fluorescence emission of the PCI telodendrimer in methanol were almost the same as those of a single ICGD molecule in methanol, indicating that the incorporation of ICGD into PCI was successful (Figures 2b and 13a).

[0111] The amphiphilic PCI telodendrimers could self-assemble into thermodynamically stable nanoparticles (PCI-NPs) with uniform size (~30 nm), which was verified by dynamic light scattering (DLS) and transmission electron micrograph (TEM) images simultaneously (Fig. 2c). Assuming π-π interactions and strong hydrophobicity, ICGD and CA could easily self-assemble into highly ordered structures to form the hydrophobic core of PCI-NPs. As shown in Fig. 13b, the UV-vis absorption of PCI-NPs in PBS is broad and slightly red-shifted compared to that of PCI telodendrimers in methanol, indicating the self-assembly of ICGD. Meanwhile, the fluorescence intensity of ICGD in PCI-NPs in PBS solution was found to be very weak (Ex: 808 nm), possibly due to the quenching effect due to aggregation, further revealing that ICGD self-assembled into highly ordered structures. This interpretation was further verified by the recovery of fluorescence upon micelle dissociation. The strong ionic detergent sodium dodecyl sulfate (SDS; 2.5 mg mL -1When ICGD (final concentration) was added to the PCI-NP PBS solution, the PCI-NPs were disintegrated and the fluorescence intensity of ICGD with a peak value at 835 nm was significantly enhanced (Figure 13c).

[0112] To improve the stability of PCI-NPs for in vivo studies, hybrid NPs with disulfide bridges were prepared by mixing a reasonable weight ratio (1:1) of cysteine-containing telodendrimers (PEG 5k CPCI-NPs were prepared by co-self-assembly of PCLC (-Cys4-L8-CA8) and PCI telodendrimers to produce CPCI-NPs. As shown in Figures 12a and 12b, eight CA and four cysteine ​​units were introduced into the backbone of PCLC telodendrimers (Mn ≈ 12000). The thiol groups on the cysteines could form disulfide bonds in the hydrophobic core of the NPs under oxidative environment, like a bundled rope, which could improve the density and stability of the NPs. As shown in DLS and TEM images (Figure 2d), under this composition design, the size of CPCI-NPs was smaller than that of PCI-NPs, i.e., 20 ± 6 nm vs. 30 ± 8 nm. This is a desirable particle size for efficient accumulation in tumors due to the enhanced penetration and retention (EPR) effect. The stability of PCI-NPs and CPCI-NPs was improved at a final concentration of 2.5 mg mL -1 The NPs were tested in the presence of NP-disrupting SDS. As shown in Figure 2e, the immediate disappearance (<10 s) of the particle size signal of PCI-NPs reflected the distinct dynamic binding-dissociation characteristics of non-crosslinked PCI-NPs. In sharp contrast, CPCI-NPs remained at a particle size of about 20 nm even in the presence of SDS, indicating the high stability of such crosslinked NPs. Furthermore, in the presence of SDS and intracellular reduced glutathione (GSH) levels (~10 mM), the particle size signal of CPCI-NPs disappeared rapidly, indicating the dissociation of CPCI-NPs after cleavage of disulfide bonds by GSH. Similar to PCI-NPs, the fluorescence of ICGD in CPCI-NPs (Ex: 808 nm) was also highly quenched. Neither GSH (~10 mM) nor SDS (2.5 mg mL -1) alone could not degrade the crosslinked NPs, so the fluorescence intensity of the CPCI-NP solution increased only slightly in SDS solution without GSH or in PBS solution with GSH. However, as shown in Figure 15d, in the presence of both SDS and GSH, the fluorescence intensity of the CPCI-NP solution with a peak value at 835 nm was enhanced 100-fold compared to the intact micellar form in PBS.

[0113] Photothermal properties of CPCI-NPs The light-to-heat conversion characteristic is 0.8Wcm -2The temperature change of CPCI-NP and PCI-NP in PBS solution under 808 nm laser irradiation at an output intensity of 1000 nm was evaluated by quantitative measurement, and polyethylene glycol (PEG)-coated gold nanorods (GNRs) were used as a control. As shown in Figure 2f, both CPCI-NP and PCI-NP solutions showed a rapid temperature increase during the first 60 s, with the temperature increasing to 64.5 °C for CPCI-NP and 56.6 °C for PCI-NP. After 300 s of continuous laser irradiation, the solution temperatures of both CPCI-NP and PCI-NP reached a stable level, and the temperature of the CPCI-NP solution (78.5 °C) was about 10.5 °C higher than that of the PCI-NP solution in ICGD with the same concentration. The maximum temperature of GNRs (51.2 °C) was similar to the maximum temperatures of other gold nanostructures reported previously. According to previously reported procedures, the photothermal conversion efficiency (η) of CPCI-NPs at 808 nm was calculated to be 30.6%, higher than that of PCI-NPs (24.3%) and GNRs (18.1%). These results indicate that CPCI-NPs have more efficient photothermal conversion than GNRs. Subsequently, the photothermal stability of CPCI-NPs and PCI-NPs was also studied by alternating heating and cooling of the NP solutions in comparison with the FDA-approved photothermal agent, indocyanine green (ICG). CPCI-NPs were found to maintain a robust photothermal efficiency even after five cycles of 808 nm laser irradiation (Figure 2g), slightly higher and much higher stability than PCI-NPs and ICG PBS solutions, respectively. Similarly, CPCI-NPs exhibited a much higher plateau temperature and faster temperature rise rate compared to PCI-NPs. These high photothermal conversion efficiencies and high stability of CPCI-NPs can be attributed to the strong π-π interactions and the dense organization of ICGDs caused by crosslinking (Figures 2c, 2d).

[0114] To further study the high photothermal conversion efficiency of CPCI-NPs, the variation of the fluorescence intensity at different concentrations of PCI-NPs and CPCI-NPs in the assembled and disassembled forms was investigated. As shown in Figures 3a and 3b, the fluorescence intensity of micellar PCI-NPs and CPCI-NPs was very weak in PBS due to the fluorescence self-quenching of ICGD in the core of the NPs. In contrast, the fluorescence intensity of PCI-NPs and CPCI-NPs solutions was found to be significantly enhanced when dissociated with SDS. Meanwhile, the photothermal conversion measurements of PCI-NPs and CPCI-NPs showed the exact opposite characteristics, i.e., high photothermal conversion efficiency in the micellar form and low photothermal conversion efficiency in the dissociated form. The compact morphology of ICGD in micellar PCI-NPs or CPCI-NPs resulted in efficient heat generation under laser irradiation at 808 nm (Figures 3c and 3d). It is noteworthy that while the fluorescence intensity of CPCI-NPs was weaker than that of PCI-NPs (e.g., 320 vs. 800 at a PCI concentration of 10 μM), the photothermal conversion efficiency (temperature) was higher than that of PCI-NPs at the same concentration in PBS (e.g., 69.6 °C vs. 58.2 °C at a PCI concentration of 10 μM, Figures 3e and 3f), indicating that the ICGD configuration of CPCI-NPs could convert excited state energy to heat energy more efficiently instead of fluorescence. These results revealed that the stable and dense micellar structure plays an important role in the thermal conversion of the photothermal conversion agent.

[0115] Considering that CPCI-NPs will be used in vivo, the tissue depth and temperature relationship of CPCI-NPs under laser irradiation at 808 nm was determined in vitro using 2 mm thick beef slices as a surrogate tissue barrier for light. Conventional ICG after complexing with TBAI was physically encapsulated in PCLC telodendrimers as a control. As shown in Figure 3g and 3h, CPCI-NPs and PCLC / TBAI&ICG-NPs were exposed to 0.8 W cm -2Laser irradiation at 808 nm through a 2 mm slice of beef for 30 seconds at 400 nm can reach 70°C and 50°C, respectively. The temperature of PCLC / TBAI&ICG NPs was found to decrease to below 40°C when the thickness of the beef was increased to 4 mm. In contrast, the temperature of CPCI-NPs could reach about 45°C even when the thickness of the beef was increased to 6 mm. Since the thickness of normal human skin and facial skin is generally less than 4 mm and 2 mm, respectively, photothermal therapy with CPCI-NPs could potentially be used to treat primary skin cancers, including melanoma. The excellent performance of this photothermal nanosystem can be attributed to its high NIR absorbance, outstanding photothermal conversion efficiency, faster heating ability and excellent photostability.

[0116] DOX loading capacity of CPCI-NPs It has been previously demonstrated that encapsulated doxorubicin can synergize the phototherapeutic effect of porphyrin-based nanocarriers. Based on the experience with telodendrimer-based micellar nanocarriers, CPCI-NPs are expected to be excellent carriers for doxorubicin (DOX) (Figure 4a). As shown in Figure 14a, the drug encapsulation efficiency of DOX by CPCI-NPs was 0.5 mg mL -1 The DOX concentration was 100% at 3 mg / mL. -1When the concentration was increased to 100 mM, the encapsulation efficiency was still over 70% and the particle size remained at about 20 nm, confirming the excellent drug loading capacity of CPCI-NPs. CPCI / DOX-NPs exhibited good serum stability in aqueous solutions at 37 °C. When 10% fetal bovine serum (FBS) was present in the solution, the particle size of CPCI / DOX-NPs did not change in a neutral environment over the 96 h experimental period (Figure 4b). Correspondingly, DOX could be controllably released from CPCI / DOX-NPs under stimulation. Due to the fluorescence quenching of the encapsulated DOX molecules, the CPCI / DOX-NP PBS solution showed a very weak fluorescence signal of DOX. However, due to the cleavage of disulfide bonds, the release of DOX was increased, and therefore the fluorescence intensity of DOX was higher in GSH (approximately 10 mM). When CPCI / DOX-NPs were in the presence of GSH (approximately 10 mM), the fluorescence intensity of DOX was 808 nm (0.8 W cm -2 The fluorescence intensity of DOX could be further enhanced when the CPCI / DOX-NPs were perturbed with laser irradiation (300 nm, 5 min) (Fig. 4c). Similar results were shown in quantitative DOX release experiments. As shown in Fig. 4d, DOX release from CPCI / DOX-NPs exhibited a typical GSH- and light-responsive release profile. In the absence of GSH and 808 nm laser irradiation, the DOX release rate in PBS was very slow (5.1% after 4 h, less than 11% after 24 h). When GSH (~10 mM) was added to the solution, the amount of DOX release increased to 30% after 24 h. It is noteworthy that DOX release always increased dramatically upon laser irradiation, and the total release of DOX could be further increased to 55% after 24 h when CPCI / DOX-NPs were exposed to GSH (~10 mM) and sequentially irradiated with 808 nm laser (3 times, 2 min each time). The enhanced release of DOX is probably due to the cleavage of S-S bonds in a reducing environment caused by GSH and the local temperature increase induced by laser-induced photothermal conversion, both of which can accelerate the diffusion of DOX from the crosslinked NPs. On-demand GSH (or N-acetylcysteine) and photoresponsive drug release can be utilized to reduce the side effects of chemotherapeutic agents on normal tissues and enhance the cytotoxic effect at the tumor site after a significant portion of CPCI / DOX-NPs has reached the tumor site.

[0117] Cellular uptake and in vitro antitumor activity of CPCI / DOX-NPs The cellular uptake and intracellular distribution of CPCI / DOX-NPs were evaluated by confocal laser scanning microscopy (CLSM) in OSC-3 oral cancer cells. Confocal fluorescence micrographs clearly showed the intracellular distribution of DOX and CPCI-NPs (Fig. 4e). After 6 h of incubation, most of the green fluorescent signal from ICGD remained in the cytoplasm. In contrast, DOX fluorescence was mainly observed in the nucleus at this time point, and the nucleus turned pink due to the overlap of red DOX and blue 4',6-diamidino-2-phenylindole (DAPI) fluorescence. This result revealed that CPCI / DOX-NPs were rapidly taken up by cancer cells, and then the encapsulated DOX could be released into the nucleus for DNA insertion in a short time.

[0118] The synergistic chemo / photothermal effect of CPCI / DOX-NPs was evaluated in OSC-3 cells by MTS assay. As shown in Figure 4f, laser-free CPCI-NPs significantly increased the activity of the CPCI-NPs at high concentrations (200 μg mL ) after 12 h of incubation. -1 ) showed negligible toxicity to cells. However, 808 nm NIR irradiation (0.8 W cm -2 When CPCI-NPs were applied (2 min, 2 min), the viability of OSC-3 cells decreased significantly with increasing CPCI-NP concentration. Cell viability was 0.01% at 200 μg mL -1 The photothermal effect of CPCI-NPs was less than 5% at a concentration of 50 μg mL, indicating that the photothermal effect of CPCI-NPs can effectively kill cancer cells. -1Further study was carried out using a concentration of CPCI-NPs of 100 μg / mL. At this low concentration of CPCI / DOX-NPs alone, only 45% of cells were killed without laser irradiation. In sharp contrast, when cells were treated with CPCI / DOX-NPs combined with 808 nm laser irradiation, more than 85% of cells were killed. This was more effective than free DOX (61% cell viability) or CPCI-NPs under 808 nm laser irradiation (46% cell viability) (Figure 5g). Live / dead cells were co-stained with DiO (green fluorescence) and propidium iodide (PI, red fluorescence). As shown in Figure 4h, after treatment with CPCI / DOX-NPs under 808 nm laser irradiation, most of the cells were apoptotic / dead and stained with red fluorescence by propidium iodide (PI), while a significant amount of live cells emitting green fluorescence could still be observed in the other control groups. The combination treatment effect by calculating the combination index was determined. The results showed excellent synergistic effects between DOX and CPCI-NPs + light in killing tumor cells in vitro with increasing DOX and CPCI-NPs concentrations (Figures 4i and 4j). Annexin FITC / PI assay was performed to clarify whether the above synergistic effects on OSC-3 cells could result in enhanced cell apoptosis. As shown in Figure 17, neither 808 nm laser irradiation nor CPCI-NPs had significant pro-apoptotic effects. In comparison, both CPCI / DOX-NPs without laser irradiation and CPCI-NPs with laser irradiation could enhance apoptosis, resulting in 27.39% and 27.31% apoptotic cells, respectively. When CPCI / DOX-NPs were used in combination with laser irradiation, the percentage of apoptotic cells further increased to 51.1%, which was consistent with the MTS results.

[0119] Biodistribution and drug delivery of CPCI / DOX-NPs After demonstrating the synergistic function of CPCI / DOX-NPs + 808 nm laser irradiation in in vitro experiments, we evaluated its efficacy in animal tumor models. To investigate the potential toxicity of CPCI-NPs, three dose levels of CPCI-NPs were intravenously administered to healthy Balb / c mice (50, 100, and 200 mg kg based on telodendrimers). -1 Blood was drawn on days 1 and 3 for serum chemistry and blood counts. White blood cells (WBC), lymphocytes, eosinophils, red blood cell distribution width (RDW), platelets and mean platelet volume (MPV) were normal on days 1 and 3 (Figure 15). Similarly, liver function tests (alanine aminotransferase, alkaline phosphatase, aspartate transaminase, bilirubin), renal panel (blood urea nitrogen, creatinine) and other blood chemistries (albumin, total protein, cholesterol, high density lipoprotein, low density lipoprotein and triglycerides) were all found to be normal (Figure 16), with no obvious hepatic or renal toxicity. It can be concluded that CPCI-NPs are highly biocompatible photothermal conversion nanoagents with no significant side effects in mice. As CPCI-NPs are cross-linked with disulfide bonds, they are expected to be relatively stable during circulation. A pharmacokinetic study comparing free ICG, ICG and TBAI coencapsulated in PCLC-cysteine ​​containing telodendrimers (PCLC / TBAI&ICG NPs), and CPCI-NPs was performed in female Sprague-Dawley rats. As shown in Figure 5a, the non-hydrophilic ICG rapidly diffused into tissues and was cleared from the circulation within 30 min after intravenous administration (t 1 / 2 = 5.22 min; AUC = 12.091 mg / L*h). After 48 h of intravenous injection, the concentration of ICG from PCLC / TBAI&ICG NPs in the blood was 9.4% of the injected dose per gram of tissue (%ID g -1 ) to (t 1 / 2 = 28.421; AUC = 677.571 mg / L*h). In contrast, approximately 20.4% of ICGD from CPCI-NP was -1 is still in the blood circulation (t 1 / 2= 41.098; AUC = 1196.274 mg / L*h), indicating high stability of ICGD and CPCI-NP.

[0120] The particle size of CPCI / DOX-NPs is approximately 20 nm (Figure 2d), which is an optimal size for tumor targeting and penetration. Due to their relatively small size, reversibly cross-linking nature and unique architecture-dependent near-infrared fluorescence properties, CPCI / DOX-NPs are particularly suitable for use as activatable optical nanoprobes for improved cancer detection through background suppression in blood, as well as accumulation and signal amplification at preferential tumor sites. Figure 4c shows the particle size distribution of CPCI / DOX-NPs (DOX: 1.25 mg kg -1 , PCI telodendrimer: 12.5 mg kg -1 and PCLC telodendrimer: 12.5 mg kg -1 Figure 5 shows near-infrared fluorescence imaging of athymic mice bearing intraepithelial (in situ) OSC-3 oral cancer after intravenous (iv) injection of ICGD. The fluorescent signal of ICGD (indicated by green fluorescent protein (GFP)) at the tumor site was found to be time-dependent and reached a peak 36 h after CPCI / DOX-NPs injection (Figure 5c). The strong fluorescence at the tumor site showed no obvious attenuation even 48 h after injection, which implied a sustained tumor accumulation of CPCI / DOX-NPs, which implied a wide time window for initiating phototherapy in vivo. Semi-quantitative biodistribution based on ex vivo imaging of major organs and tumors collected 48 h after injection showed high tumor uptake of CPCI / DOX-NPs (Figures 5d and 5f). Meanwhile, to further verify the tumor accumulation of CPCI / DOX-NPs, tumor tissue slices were observed by fluorescence microscopy. As shown in Figure 5e, highly consistent fluorescent signals of ICGD (green) and DOX (red) appeared simultaneously in the tumor area.

[0121] The tissue distribution of DOX over time in nude mice bearing orthotopic OSC-3 oral cancer after tail vein administration of CPCI / DOX-NPs was determined. At 24 h, 48 h, and 5 days after injection, DOX concentrations in tumors and normal organs were measured by high pressure liquid chromatography mass spectrometry (HPLC-MS). As shown in Figure 5g, 24 h after CPCI / DOX-NPs administration, significant accumulation of DOX in tumors, liver, and even kidneys was observed. However, the accumulated concentration of DOX in liver and kidneys disappeared rapidly over time, especially after 5 days. More excitingly, the DOX concentration at the tumor site was up to about 2 μg per g of tissue even 5 days after injection of CPCI / DOX-NPs. This may explain the antitumor effect shown below. DOX is commonly used to treat many different cancers, and cardiotoxicity and bone marrow suppression are dose limiting toxic side effects of this very important drug. Fortunately, Figures 5e and 5g show that the fluorescence intensity of ICGD and DOX concentration in the heart are very low, indicating low accumulation of CPCI / DOX-NPs in the heart and low DOX release, a crucial attribute that is necessary for the successful future clinical application of CPCI / DOX-NPs.

[0122] Synergistic in vivo photothermal / chemotherapeutic effects based on CPCI / DOX-NPs Encouraged by the excellent biodistribution and in vitro antitumor activity of the CPCI / DOX-NP system, we next conducted a therapeutic efficacy study in athymic mice bearing orthotopic oral cancer. First, the in vivo photothermal effect of CPCI / DOX-NP was investigated. 24 hours after systemic administration of PBS, PCI-NP, CPCI-NP, or CPCI / DOX-NP, 0.8 Wcm was administered to the tumor area of ​​living mice. -2The tumor sites were irradiated with an 808 nm laser with an output intensity of 1000 Hz for 2 min. As shown in Figures 6a and 6b, upon laser irradiation, the average temperature of the tumor regions in both the CPCI-NP and CPCI / DOX-NP groups increased by about 50 °C. This was about 1.37 times higher than that of the PCI-NP (ΔT ≒ 36.5 °C) group and 8.92 times higher than that of the PBS (ΔT ≒ 5.6 °C) group. Such a high temperature increase was more than enough to cause irreversible damage to the cancer cells. It is noteworthy that it only took 45 seconds for the tumor sites in the CPCI-NP or CPCI / DOX-NP groups to rapidly heat up above 40 °C. This was consistent with the fast heating rate in the in vitro study. These results clearly demonstrated that CPCI / DOX-NPs could efficiently absorb NIR light energy and convert it into local heat in vivo for photothermal therapy.

[0123] The therapeutic effects of these photothermal agents were investigated in nude mice bearing orthotopic OSC-3 cell oral cancer. Mice were randomly divided into five groups: (1) PBS, (2) free DOX, (3) PCI-NPs, (4) CPCI-NPs, and (5) CPCI / DOX-NPs. Near-infrared laser irradiation (808 nm, 0.8 W cm -2 The experimental design of the animals is shown in Figure 6c. 3 When the tumor reached 100 nm, CPCI / DOX-NPs were injected intravenously into the tail vein, and the tumor area was then exposed to 808 nm laser irradiation for 2 min (0.8 Wcm) at 24 and 48 h (i.e., days 2 and 3) after injection. -2) exposure. The same treatment regimen (photothermal agent followed by light irradiation) was then repeated on days 7, 8, and 9. As shown in Figure 6d, the PCI-NP, CPCI-NP, and CPCI / DOX-NP groups showed satisfactory antitumor effects during the first 10 days of treatment. However, in both the PCI-NP and CPCI-NP groups, tumors began to regrow at the primary site after day 10. In contrast, complete tumor remission was achieved in animals that received CPCI / DOX-NP + laser irradiation, verifying the synergistic therapeutic efficacy of photothermal / chemotherapy. Figure 5e shows the photographs of mice treated with CPCI / DOX-NP + laser irradiation. No tumors were found on day 28. Moreover, there was no significant change in body weight after treatment (Figure 18). Histological analysis further confirmed that most tumor cells were destroyed with the treatment of CPCI / DOX-NPs + 808 nm laser irradiation, with a much lower level of nuclear polymorphism and lower cancer cell density in tumor sections than the other groups ( Figure 6 f).

[0124] Synergistic effects of CPCI / Imiquimod-NP-based in vivo photothermal / immunotherapy The experimental results mentioned above clearly show that CPCI / DOX-NPs are a highly effective phototherapy system for localized treatment when the treatment area has access to light. However, cancer is often accompanied by recurrence and metastasis, and the majority of cancer deaths are caused by metastasis. In the past few years, immunotherapy with checkpoint blockade antibodies has proven clinically very promising, but the response rates vary greatly depending on the tumor type. Imiquimod is an immune response modifier that binds to Toll-like receptor 7 and has been approved by the US Food and Drug Administration (FDA) for the topical treatment of genital warts, superficial basal cell carcinoma, and actinic keratosis. It is too toxic to be administered systemically. Due to the excellent encapsulation ability of CPCI-NPs, imiquimod has a very high drug encapsulation capacity (imiquimod concentration: 0.5 mg mL ) while maintaining the desired nanoparticle size of 22 ± 4 nm for tumor accumulation by the EPR effect. -1) could be loaded into the core of CPCI-NPs (Figure 7b). We investigated whether the new CPCI / imiquimod-NPs-based photothermal / immunotherapy could be combined with PD-1 checkpoint blockade therapy to enhance the anticancer synergistic therapeutic effect against metastatic tumors (Figure 6a).

[0125] The design of the animal experiment is shown in Figure 7c. An artificial mimic of metastatic tumors (bilateral breast tumor model) was developed by orthotopically injecting 4T1 cells into both the left and right mammary fat pads of the same immunocompetent mice. The left tumor was designated as the primary tumor to be treated with light, and the right tumor was inoculated as a distant tumor or "metastatic" tumor without light treatment. Mice were randomly divided into seven groups (n=6 per group): (1) PBS (untreated), (2) CPCI / Imiquimod-NP for two cycles of treatment, (3) CPCI-NP combined with laser irradiation for two cycles of treatment, (4) CPCI / Imiquimod-NP combined with laser irradiation for two cycles of treatment, (5) CPCI / Imiquimod-NP+anti-PD-1 for two cycles of treatment, (6) CPCI / Imiquimod-NP+anti-PD-1 combined with laser irradiation for one cycle of treatment, (7) CPCI / Imiquimod-NP+anti-PD-1 combined with laser irradiation for two cycles of treatment. In group 7, tumor volume was approximately 50 mm 3 When the tumor reached a tumor volume of 100 μg / mL, the CPCI / imiquimod-NPs solution was injected via the tail vein. 24 h after injection, the primary tumor was locally exposed to 808 nm laser irradiation for 2 min (0.8 W cm -2). Then, on day 2, the mice were intraperitoneally (ip) injected with anti-PD-1 (α-PD-1) at a dose of 200 μg per mouse. Then, the second cycle administration (intravenous injection of CPCI / imiquimod-NPs, tumor local laser irradiation, and intraperitoneal injection of α-PD-1) was repeated once on days 6, 7, and 8. The growth of primary and distant tumors in different groups was measured by calipers every other day. As shown in Figure 7d, primary tumors in all groups that were locally irradiated with 808 nm laser were nearly eliminated by CPCI-NP-based early-stage photothermal therapy. Due to the lack of further treatment such as a second laser irradiation, or the lack of imiquimod or α-PD-1 immune effects, slight tumor recurrence occurred in control groups 3, 4, and 6 after laser irradiation. It is noteworthy that group 5 (CPCI / Imiquimod-NP+α-PD-1) delayed the inhibition of primary tumor growth to some extent, but failed to eliminate the tumor without laser irradiation. In sharp contrast, two cycles of combination therapy of CPCI / Imiquimod-NP and α-PD-1 (group 7) could significantly eliminate the primary tumor without recurrence within the first 16 days, indicating that such non-specific combination immunotherapy can be effective in cancer treatment.

[0126] Meanwhile, as shown in Fig. 7e, in mice whose primary tumors were ablated by CPCI / Imiquimod-NPs-based PTT, the growth of their distant tumors was also partially delayed. Especially in group 7, their distant tumors showed almost complete disappearance, achieving a much better effect than that obtained in group 5 (CPCI / Imiquimod-NPs and α-PD-1 treatment, but without PTT of primary tumors). It was verified that PTT for primary tumors also induced obvious inhibitory effects on distant tumors. Representative photographs of the volume changes of primary and distant tumors in mice treated with group 7 were shown (Fig. 7f). As another control, the immune response induced by group 4 (α-PD-1 blockade therapy together with CPCI / Imiquimod-NPs-based PTT of primary tumors) was much better than that of group 3 (without imiquimod), indicating that the use of imiquimod plays an important role for those NPs to induce a strong immune response. Finally, the immune response induced by CPCI / Imiquimod-NPs-based PTT in primary tumor group 4 (without α-PD-1) or group 6 (one cycle of α-PD-1 treatment only) could only inhibit the growth of secondary tumors at some time in the early days. This revealed that the α-PD-1 blockade treatment strategy played a central role in continuously killing tumor cells by activating T cells to recognize tumor cells. The mechanism of the synergistic antitumor effect caused by CPCI / Imiquimod-NPs-based PTT combined with α-PD-1 therapy was further investigated. In the experiment, mice were divided into four groups for one cycle of treatment (group 1: PBS, group 2: CPCI / Imiquimod-NPs, group 3: CPCI / Imiquimod-NPs + laser irradiation, group 4: CPCI / Imiquimod-NPs + laser irradiation + anti-PD-1, n=6 per group). Then, the mice were sacrificed and the tumor residual tissues were collected bilaterally for evaluation by H&E staining, RT-PCR and IHC. H&E staining directly showed large areas of cell destruction and apoptosis in the primary tumors in groups 3 and 4, and distant tumors also shrank due to the response of the immunotherapy effect, which was in clear contrast to the control groups 1 and 2 (Figure 7g).Imiquimod is a strong TLR7 agonist and can activate natural killer cells, macrophages and B lymphocytes through TLR-7-secreted cytokines (IFN-α, IL-6 and TNF-α), further promoting the activation of immune responses. IFN-α, TNF-α, IL4, IL6 (cellular immune-related markers) and IL-12 (natural immune marker) gene expression by RT-PCR was evaluated. As shown in Figure 7h, the relative expression of these immune markers in the primary tumor side with laser irradiation was higher than that without laser irradiation, verifying that PTT can induce a strong tumor-specific immune response by producing tumor-associated antigens from resected tumor cell residues. Furthermore, the relative expression of these immune markers in distant tumors indicates the importance of photoirradiation of primary tumors (groups 3 and 4). Cytotoxic T lymphocytes (CD8. + ) can directly kill targeted cancer cells, while CD3 + and CD4 + Helper T cells may also play an important role in regulating adaptive immunity. + , CD3 + , CD4 + As shown in Figure 7i, the tumor cell counts in the treated primary tumors (groups 3 and 4) were significantly lower due to the effect of PTT treatment. Furthermore, the CD8 + , CD3 + , CD4 + and PD-1 T cells were found much more frequently in groups 3 and 4 (imiquimod and phototherapy combination groups) than in the PBS and imiquimod only groups. These results clearly demonstrate that systemic administration of CPCI / imiquimoid-NPs and anti-PD-1 checkpoint blocking antibodies in combination with local PTT is highly effective against local and distant metastatic tumors. This therapeutic concept is highly translatable and can be tested in clinical trials.

[0127] Immune memory effect To evaluate the immune memory induced by synergistic photothermal immunotherapy (CPCI / Imiquimod-based PTT combined with α-PD-1), secondary 4T1 tumors were inoculated into both the primary and distant sides 50 days after removal of the first implanted tumor. Mice of the same age injected with PBS served as controls. Similarly, α-PD-1 was intraperitoneally injected twice (200 μg per mouse each time) into the two groups of mice on days 67 and 73 (Figure 8a). As shown in Figures 8b and 8c, tumor volume rapidly increased in the PBS group even with injection of α-PD-1. In sharp contrast, the synergistic treatment strategy with CPCI / Imiquimod-NPs-based PTT given in combination with α-PD-1 could significantly delay the growth of tumors re-inoculated on both sides, indicating that this strategy has an immune memory effect via activation of the innate and adaptive immune systems. Unfortunately, this strategy only delayed tumor growth but could not completely suppress tumor recurrence. This is likely due to a lack of sufficient tumor-associated antigens produced during the initial treatment.

[0128] Discussion Near-infrared photothermal conversion agents (PTCAs) used in photothermal therapy have been investigated for many years and applied to various tumor models, but there are still great challenges in improving their photothermal conversion efficiency and stability, including rapid excretion from the kidney or metabolism in vivo by the liver and degradation / fading under prolonged laser irradiation. Therefore, there is an urgent need to develop a new generation of highly stable and high-performance PTCAs for photothermal cancer therapy. It is clear from previous and current studies that cytotoxic chemotherapeutic agents such as doxorubicin can synergize the antitumor effect of PTT. Since the majority of cancer-related deaths are due to metastasis, there is a great need to develop effective systemic therapies for metastatic disease. Immunotherapy, especially checkpoint blockade therapy with monoclonal antibodies, has emerged in recent years as a very promising approach to treat clinical cancer. Such therapies can mobilize the patient's own immune system to fight cancer. Recently, it has been reported that PTT can also generate antitumor immunological effects by generating tumor-associated antigens from excised tumor cell residues to improve antitumor immunity.

[0129] Herein, the development of novel telodendrimer-based high-performance photothermal nanoparticles (CPCI-NPs) is reported, which can not only function as an excellent PTCA but also as a convenient and efficient controlled drug delivery system for cancer treatment. The covalent attachment of ICGD to telodendrimers combined with interpolymer cross-linking by disulfide bonds allows CPCI-NPs to assemble a high density of ICGD, and the π-electron interactions ensure high photothermal conversion efficiency and photostability after self-assembly. The photothermal conversion efficiency of CPCI-NPs remained high (above 70 °C) after five cycles of laser irradiation. The temperature of the CPCI-NP solution was reduced by 60 s of irradiation (808 nm, 0.8 W cm). -2It is noteworthy that the photothermal activity of ICGD reached a nearly maximum immediately after 30 min. This suggests that the unique configuration of ICGD contributes to this desirable photothermal property. The hybrid telodendrimers containing hydrophobic ICGD and facial amphiphilic cholic acid units provided an ideal platform for encapsulation and delivery of chemotherapeutic or immunomodulatory agents. For example, CPCI / DOX-NPs have an ideal small particle size (~20 nm), tend to accumulate at the target tumor site, and penetrate into deep tissues for deep treatment after longer blood circulation (maximum concentration of CPCI / DOX-NPs at the tumor site after 36 h). The presence of disulfide bridges ensures the stability of NPs and limits the premature release of DOX in blood circulation, while DOX can be controllably released from NPs under the reducing environment of cells, under 808 nm laser irradiation (Figure 4f), or on-demand release by administration of N-acetylcysteine.

[0130] Using an orthotopic oral cancer xenograft model showed that photothermal / chemotherapy combination with CPCI / DOX-NPs was highly effective using laser irradiation 24 and 48 hours after administration of the nanoparticles. Since a significant portion of CPCI / DOX-NPs remains at the tumor site for at least 5 days, it is conceivable that phototherapy can be repeated multiple times over a 5-day period. Such a treatment approach would allow for continuous phototherapy while the tumor regresses, and thus reach deeper tumors beyond the surface 5 mm. ICGD was selected for the following reasons: (1) it has a long absorption wavelength (808 nm) and therefore a deep light penetration for phototherapy, and (2) ICG with a fluorescence emission wavelength of 808 nm has already been used in the clinic as an optical imaging probe for several decades. Thus, CPCI / DOX-NPs can not only function as an excellent PTCA, but can also be used as a tumor targeting probe for image-guided surgery. In addition, there is great clinical potential for use in intraoperative photothermal therapy when the tumor margins and tumor bed are easily accessible to phototherapy in the operating room.

[0131] In addition to local therapy, CPCI-NPs can also be used for systemic immunotherapy, especially when loaded with immunomodulators such as the FDA-approved imiquimod. When encapsulated with CPCI-NPs, the toxicity of intravenous imiquimod is much lower, allowing systemic administration of imiquimod to all tumor sites via the EPR effect. Localized light irradiation of superficial tumors or tumors accessible to endoscopy is expected to generate tumor-associated antigens and sensitization of immune cells locally, which subsequently disseminate to other metastatic tumor sites and cause abscopal antitumor effects. Such photoimmunotherapy is expected to be highly effective when combined with checkpoint blockade antibodies. This concept was tested in a 4T1 syngeneic orthotopic breast cancer model. Upon NIR-induced photothermal ablation of primary tumors after systemic injection of CPCI / imiquimod-NPs, the released tumor-associated antigens, especially with the help of anti-PD-1 checkpoint blockade therapy, demonstrate a strong immune response, activating many antigen-specific T cells, while inhibiting the activity of immunosuppressive regulatory T cells (Tregs). This can attack distant tumor cells remaining in the mouse's body. Separate experiments show that this combined photoimmunotherapy strategy can significantly delay tumor formation and slow tumor growth when treated mice are rechallenged by implantation of new 4T1 tumor cells.

[0132] In summary, a new generation of PTCAs based on co-assembled binary telodendrimers exhibited excellent photothermal conversion efficiency, heating capability and stability for photothermal therapy. Furthermore, it can be utilized as a multifunctional nanoplatform combining chemotherapy for the synergistic treatment of in situ oral cancer and immunotherapy for the synergistic treatment of metastatic breast cancer, respectively. This highly promising nanoplatform shows great potential for clinical translation against many different tumor types. It is expected that their efficacy can be further improved by introducing tumor-targeting ligands into the nanocarriers.

[0133] method material Monomethyl-terminated poly(ethylene glycol) monoamine (MeO-PEG-NH2, Mw: 5000 Da) was purchased from Rapp Polymere (Germany). 4-Carboxyphenylboronic acid, 4-carboxyphenylboronic acid pinacol ester, 3-carboxy-5-nitrophenylboronic acid, and 3-carboxy-5-nitrophenylboronic acid pinacol ester were obtained from Combi-Blocks (San Diego, CA). (Fmoc)lys(Boc)-OH, (Fmoc)Lys(Fmoc)-OH, (Fmoc)Cys(Trt)eOH, and (Fmoc)Ebes-COOH were purchased from AnaSpec Inc. (San Jose, CA). All other chemicals were purchased from Sigma-Aldrich (St. Louis).

[0134] Synthesis of indocyanine green derivatives (ICGD) Briefly, 1,1,2-trimethylbenz[e]indole (I) and iodoethane (1.5 eq.) were reacted in acetonitrile solution at 90° C. under reflux for 3 days. Ether was then added to generate 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-ium (product II). The crude product (II) was then added to N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (1 eq.) in acetic anhydride. The mixture was heated to 100° C. for 1.5 h, cooled to RT, and added to deionized water. After filtration, product (III) 3-ethyl-1,1-dimethyl-2((1E, 3E, 5E)-6-(N-phenylacetamido)hexa-1,3,5-trien-1-yl)-1H-benzo[e]indol-3-ium was obtained. Further, 1,1,2-trimethylbenz[e]indole (I) and 5-bromovaleric acid (1.2 equiv.) of potassium iodide were reacted in acetonitrile solution under heating reflux at 100 °C for 5 days. Then, ether was added to generate 3-(4-carboxybutyl)-1,1,2-trimethyl-1H-benzo[e]indol-3-ium (IV). Finally, the crude IV was added to III in pyridine. The mixture was heated at 40 °C for 30 min and concentrated in vacuum to obtain the final product. The final crude product was purified twice on a silica gel column to obtain ICGD.

[0135] Synthesis of telodendrimers The first representative ICGD / CA telodendrimer (PEG 5k-ICGD4-CA4, abbreviated as PIC), was synthesized from MeO-PEG-NH2 via solution-phase condensation reaction using stepwise peptide chemistry according to published methods (Li et al., Biomaterials 2011, 32(27), 6633-6645). Briefly, (Fmoc)Lys(Fmoc)-OH (3 equiv.) was coupled to the NH2 terminus of PEG using DIC and HOBt as coupling reagents until a negative Kaiser test result was obtained, indicating completion of the coupling reaction. The PEGylated molecule was precipitated by adding cold ether and then washed twice with cold ether. The Fmoc group was removed by treatment with 20% (v / v) 4-methylpiperidine in dimethylformamide (DMF), and the PEGylated molecule was precipitated and washed three times with cold ether. The white powder precipitate was dried under vacuum, and one coupling of (Fmoc)Lys(Fmoc)-OH and one coupling of (Fmoc)lys(Boc)-OH were performed to generate the third generation dendritic polylysine terminated with four Boc and Fmoc groups at one end of PEG, respectively. CA NHS ester and ICGD were attached to the ends of the dendritic polylysine after removing Fmoc with 20% (v / v) 4-methylpiperidine and Boc groups with 50% (v / v) trifluoroacetic acid in dichloromethane, respectively. The telodendrimer solution was filtered and then separated by dialysis against water with a MWCO of 3.5 KDa. Finally, the telodendrimers were lyophilized.

[0136] The second thiolated telodendrimer (PEG 5kThe dendritic polylysine (designated as -Cys4-L8-CA8 and abbreviated as PCLC) was synthesized according to USPN 10,106,650 B2. Briefly, a third generation dendritic polylysine terminated with four Boc and Fmoc groups at one end of PEG was first synthesized according to the above method. After removing the Boc groups with 50% (v / v) trifluoroacetic acid (TFA) in dichloromethane (DCM), (Fmoc)Cys(Trt)eOH, (Fmoc)Ebes-OH and cholic acid NHS ester were stepwise coupled to the ends of the dendritic polylysine. The Trt groups on the cysteines were removed by TFA / H2O / ethanedithiol (EDT) / triethylsilane (TIS) (94:2.5:2.5:1, v / v) to generate the PCLC thiolated telodendrimers. The thiolated telodendrimers were recovered from the mixture by three cycles of dissolution / reprecipitation with DMF and ether, respectively. Finally, the thiolated telodendrimers were dissolved in acetonitrile / water and lyophilized.

[0137] The molecular weights of the PIC and PCLC telodendrimers were collected on an ABI 4700 MALDI-TOF / TOF mass spectrometer (linear mode) using R-cyano-4-hydroxycinnamic acid as the matrix. Monodisperse mass traces of the telodendrimers were detected, and the molecular weights of the telodendrimers from MALDI-TOF MS were almost identical to the theoretical values. 1 HNMR spectra were recorded on an Avance 600 nuclear magnetic resonance spectrometer (Bruker) using CDCl3 as the solvent.

[0138] Preparation of CPCI-NPs PIC NPs were formed by dissolving 10 mg of PIC telodendrimers in 1 mL of PBS and then sonicating for 10 min. To prepare CPIC NPs, 10 mg of PCLC telodendrimers and 10 mg of PCI telodendrimers were dissolved in 1 mL of PBS to form micelles and then sonicated for 10 min. The thiol groups on the telodendrimers were oxidized by air to form disulfide bonds.

[0139] Preparation of CPCI / DOX-NPs Hydrophobic drugs such as DOX or imiquimod were loaded into CPIC NPs by a solvent evaporation method. Before encapsulating DOX into NPs, DOX·HCl was stirred with 3 molar equivalents of triethylamine in chloroform (CHCl3) / methanol (MeOH; 1:1, v / v) to remove HCl from DOX·HCl. In total, 10 mg of PCI telodendrimer and 10 mg of PCLC telodendrimer along with different amounts of neutralized DOX were first dissolved in CHCl3 / MeOH, mixed, and evaporated on a rotavapor to obtain homogenous dry polymer films. The films were reconstituted in 1 mL of PBS and then sonicated for 30 min to disperse the sample films in the nanoparticle solution. Finally, the nanoparticle solution was filtered through a 0.22 mm filter to sterilize the samples. To determine the amount of drug, the drug-loaded nanoparticles were diluted with DMSO (NP solution / DMSO, 1:9, v / v) to dissociate the nanoparticles. The drug loading was analyzed by an HPLC system (Waters). Here, the calibration curve was obtained using a series of drug / DMSO standard solutions of various concentrations. Imiquimod-loaded CPIC NPs were prepared in a similar manner, except that 3 molar equivalents of triethylamine were not used.

[0140] Nanoparticle characterization The size and size distribution of the nanoparticles were measured by DLS instrument (Microtrac). The concentration for DLS measurements was set at 1.0 mg mL -1 The nanoparticles were dissolved in water and kept at 20° C. All measurements were carried out at 25° C. The morphology of the nanoparticles was observed with a Philips CM-120 TEM. Briefly, the nanoparticles were dissolved in water (1 mg mL -1Toledendromer) was deposited on a copper grid, stained with phosphotungstic acid for 2 s, and measured at room temperature. The size of the particles was analyzed using TEM software (Digital Micrograph, Gatan Inc). The UV-vis-NIR absorption spectra of the NPs were measured on a Genesys 10S UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA). The fluorescence signal of the NPs was measured by fluorescence spectroscopy (SpectraMax M2, Molecular Devices, Suunyvale, CA, USA).

[0141] Stability of nanoparticles in SDS and human plasma Stability studies were performed to monitor the changes in fluorescence and particle size of PCI-NPs and CPCI-NPs in the presence of SDS, which has been reported to be able to efficiently disassemble polymeric micelles. Aqueous solutions of the nanoparticles (1.5 mg mL -1 ) in SDS solution (7.5 mg mL -1 The final SDS concentration was 2.5 mg / mL. -1 and the micelle concentration is 1.0 mg mL -1 The solution was kept at 4°C for 10 min. The fluorescence signal of the solution was measured by a fluorescence spectrometer. The size and size distribution of the nanoparticle solution was monitored by DLS at predefined time intervals. The stability of the micelles was also evaluated in the presence of GSH (10 mM) along with SDS. The stability of DOX-loaded CPCI / DOX-NPs was further studied in 10% (v / v) plasma from healthy human volunteers. The mixture was incubated at physiological body temperature (37°C) and then sized at predefined time intervals up to 96 h.

[0142] Photothermal effect of nanoparticles 1 mL of PCI-NP (ICGD dose 30 μg mL -1 ), CPCI-NP (ICGD dose 30μgmL -1 ) and polyethylene glycol (PEG)-coated gold nanorods (GNR concentration: 18 μg mL -1) were added into a 4 mL transparent quartz vial. The solution was irradiated with a fiber-coupled continuous-wave semiconductor diode laser (808 nm, Beijing Viasho Technology Co., Ltd., China). The power density was 0.8 W cm -2 The temperature was monitored by a thermal infrared imaging camera (Flir C2, USA). To investigate the photostability of CPCI-NPs, 1 mL of CPCI-NPs (30 μg mL -1 ICGD concentration at 30 μg mL -1 ICGD concentration at 30μgmL -1 The aqueous solution was irradiated with an 808 nm laser for 5 minutes and then cooled to room temperature.

[0143] In vitro penetration depth and thermal effects of nanoparticles To investigate the relationship between the heating effect of CPCI-NPs under laser irradiation at 808 nm and the tissue penetration depth, an in vitro simulation experiment was first designed. The experiment was divided into two groups: (1) CPCI-NPs and (2) PCLC / TBAI&ICGNP. Group (2) was prepared in the following manner. The complex of free ICG and tetra-n-butylammonium iodide (TBAI) via electrostatic interactions could be physically encapsulated in a PCLC toredendrimer, named PCLC / TBAI&ICGNP. Hotpot beef was chosen as the simulation tissue and cut into square pieces (thickness of each piece: 2 mm). 100 μL of CPCI-NPs (30 μg mL -1 ICGD concentration in TBAI) or PCLC / TBAI&ICG NP (free ICG concentration: 30μgmL -1 ) solution on the surface of the beef and then irradiated with an 808 nm laser (0.8 Wcm -2 A thermal pressure of 1000 psi (2 min) was applied underneath the device. The temperature and thickness of the beef pieces were recorded by a thermal infrared camera.

[0144] DOX release from CPCI / DOX-NPs The in vitro DOX release profile from CPCI / DOX-NPs was further measured by dialysis. The drug release experiments were divided into three groups (n = 3): (1) PBS, (2) GSH (10 mM), (3) NIR irradiation (808 nm laser, 0.8 W cm -2 ) together with GSH (10 mM). First, 500 μL of CPCI / DOX-NP solution (DOX concentration 1 mg mL -1 ) was transferred to a dialysis bag and dialyzed against DI water. All groups were initially evaluated for 4 hours at 37°C with shaking. Group (2) was then added with GSH. Group (3) was added with GSH and irradiated under 808 nm laser three times at the appropriate time points. For all groups, 10 μL of the solution was removed at the desired time points and added to one bottle containing 90 μL of DMSO. The released DOX content was analyzed by HPLC system (Waters), where a calibration curve was obtained using a series of drug / DMSO standard solutions with different concentrations. Data were reported as the average percentage of DOX cumulative release for each of the three samples.

[0145] In vitro cell experiments In vitro cytotoxicity of samples, cellular uptake studies and combination therapy were evaluated in human OSC-3 oral cancer cell line purchased from American Type Culture Collection (ATCC). Cells were cultured for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 using DMEM supplemented with 10% FBS and 1% penicillin. Samples were then added to each well and cells were incubated for another 24 h at 37 °C. After incubation, relative cell viability was measured by MTS assay. To evaluate cellular uptake, OSC-3 cells were seeded in 96-well plates and incubated with CPCI / DOX-NPs (DOX concentration: 25 μg mL -1 ) for an additional 6 h. Cellular uptake was determined by confocal imaging after 30 min of DAPI staining. For in vitro chemotherapy / photothermal combination therapy, OSC-3 cells were seeded in 96-well plates (n=3) and incubated with the various samples for 4 h, then illuminated with an 808 nm NIR laser (0.8 Wcm -2, 2 min). After that, the cells were incubated for another 24 h at 37 °C, and MTS reagent was added to each well. Then, the relative cell viability was measured by MTS assay. The percentage of cell viability represents the drug effect, and 100% means that all cells survived. The cell viability was calculated using the following formula: Cell viability (%) = (treated OD490nm / blank control OD490nm) × 100%. For in vitro cell apoptosis experiments, OSC-3 cells were incubated with various samples (DOX concentration: 25 μg mL -1 and 808 nm NIR laser (0.8 W cm -2 The cells were co-cultured with PI / DioFITC for 8 h. The cells were washed twice and replaced with fresh medium. Then, the cells were co-cultured with PI / DioFITC for 30 min in a 12-well plate. Cell apoptosis was monitored using a fluorescent microscope. Representative FCM analysis scattergrams of PI / Annexin V showed apoptosis in the four groups.

[0146] In vivo blood clearance kinetics A blood sampling catheter was implanted in the jugular vein of female Sprague-Dawley rats (Harland, Indianapolis, IN, USA). Free ICG, PCLC / TBAI&ICG NPs, and CPCI-NPs with the same ICG concentration were administered at 5 mg kg -1 The dose was injected through the catheter according to body weight (n=3 in each group). All blood samples were collected from the catheter at different time points. The ICG concentration was measured by absorbance at 808 nm using a microplate reader (SpectraMax M2).

[0147] Animal models: All animal experiments followed protocol No. 19724 approved by the University of California, Davis, Animal Use and Care Management Advisory Committee. In situ OSC-3 oral cancer model: 6-8 week-old female nude mice purchased from Harlan (Livermore, CA, USA). The orthotopic oral cancer model was implanted on the lips, which was established for the first time. Each female nude mouse was 1 × 10 6Mice bearing OSC-3 oral tumors were transplanted by injecting 1000x1000 cells into the follicular cavity of the follicular cavity. 3 4T1 breast cancer model: Female Balb / c mice (8-10 weeks) were purchased from Harlan (Livermore, CA, USA). 4T1 cells (1 × 10) suspended in an 80 μL mixture of PBS and Matrigel (1:1 v / v) were then cultured in a 10-well plate. 6 ) was injected into the fourth mammary fat pad. Mice bearing 4T1 tumors were cultured until the tumor volume reached approximately 50 mm 3 was treated when

[0148] In vivo / ex vivo fluorescence imaging Nude mice bearing intraepithelial OSC-3 oral cancer were subjected to tail vein injection of CPCI / DOX-NPs at a dose of 7 mg ICGD per kg body weight. Mice under isoflurane anesthesia were imaged using an in vivo fluorescence imaging system (Carestream In-Vivo Imaging System FX PRO, USA) at pre-designed time points. 48 h after NP injection, all animals were sacrificed and their organs of interest were excised for ex vivo imaging.

[0149] In vivo cancer treatment Mice bearing in situ OSC-3 oral cancer were treated with PBS, free DOX (2.5 mg / mL -1 ), PCI-NP (PCI Telodendrimo 25 mg kg -1 ; ICGD 7mgkg -1 ), CPCI-NPs (total telodendron 50 mg kg -1 ; ICGD 7mgkg -1 ), CPCI / DOX-NPs (total telodendron 50 mg kg -1 ; DOX 2.5mgkg -1 , ICGD 7mg kg -1 ) was injected. 24 and 48 hours later, the tumor was locally irradiated twice with a laser under general anesthesia (0.8 Wcm -2The entire tumor was covered with the beam spot at 1000 nm for 2 min. Mice were treated once a week for 2 cycles. A thermal camera (FLIR) was used to record the tumor surface temperature. Tumor volume was measured twice a week by caliper, where "w" and "l" are the width and length of the tumor. Treatment continued for 30 days. Mice were sacrificed when their body weight was reduced by 20 percent or when tumor growth affected drinking and touching. Mice bearing 4T1 tumors were treated with PBS, CPCI / Imi, CPCI+L, CPCI / Imi+L, CPCI / Imi+α-PD-1, CPCI / Imi+L+α-PD-1 and CPCI / Imi+L+α-PD-1 (1 cycle) (ICGD 7 mg kg -1 , imiquimod 1.25 mg kg -1 The mice were randomly divided into seven groups (n=6) for treatment with anti-PD-1 (IP injection of 200 μg per mouse). 3 Treatment was administered when tumor volume reached 100%. Tumor size was measured twice a week and calculated using the following formula: volume = 0.5 × L × W 2 .

[0150] Histology research Mice bearing OSC-3 oral cancer were sacrificed after two cycles of treatment after injection. Tumor tissues were fixed and embedded in paraffin. Hematoxylin and eosin (H&E) stained slides were prepared by BBC Biochemical (Mount Vernon, WA) and observed using a microscope.

[0151] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be made within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between this application and a reference provided herein, this application shall control.

Claims

1. Formula (I) 【Chemistry 1】 wherein PEG is a polyethylene glycol (PEG) polymer, said PEG polymer having a molecular weight of 1-100 kDa; each X is a branched monomer unit; Each R 1 is independently cholic acid or a cholic acid derivative, and Each R 2 are independently cyanines) Compound.

2. The compound of claim 1 , wherein X is a diamino carboxylic acid.

3. 3. The compound of claim 2, wherein each diaminocarboxylic acid is independently selected from the group consisting of 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid, and 5-amino-2-(3-aminopropyl)pentanoic acid.

4. 3. The compound of claim 2, wherein each diamino carboxylic acid is lysine.

5. Each R 1 are independently selected from cholic acid, (3α,5β,7α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid (CA-4OH), (3α,5β,7α,12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid (CA-5OH) and (3α,5β,7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholic acid (CA-3OH-NH 2 5. The compound according to claim 1, wherein

6. Each R 1 The compound according to any one of claims 1 to 5, wherein is cholic acid.

7. Each R 2 is independently cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, cyanine 7.5, indocyanine green, or structure (S1): 【Chemistry 2】 The compound according to any one of claims 1 to 6, which is an indocyanine green derivative having the formula:

8. Each R 2 The compound of claim 7, wherein is a cyanine having the structure (S1).

9. The PEG has a molecular weight of 5 kDa; each X is a lysine; Each R 1 is cholic acid, and Each R 2 9. The compound according to claim 7 or 8, wherein is a cyanine having the structure (S1).

10. A nanoparticle comprising a plurality of first conjugates, each first conjugate being independently a compound according to any one of claims 1 to 9; The plurality of conjugates self-assemble in an aqueous medium to form a nanoparticle, such that a hydrophobic core is formed in the interior of the nanoparticle, wherein the PEG of each conjugate self-assembles on the exterior of the nanoparticle.

11. Further comprising a plurality of second conjugates, each second conjugate independently having the formula (II): 【Chemistry 3】 wherein PEG is a polyethylene glycol (PEG) polymer, said PEG polymer having a molecular weight of 1-100 kDa; each X is a branched monomer unit; each Y is a thiol-containing crosslinkable group; each L is a linking group, and Each R 1 and are independently cholic acid or a cholic acid derivative.

12. The nanoparticles according to any one of claims 10 to 11, wherein each first conjugate is a compound according to claim 9.

13. Each second conjugate is a compound of formula (II), wherein: The PEG has a molecular weight of 5 kDa; each X is a lysine; each Y is a cysteine; Each L has the formula: 【Chemistry 4】 and Each R 1 The nanoparticle of claim 11 or 12, wherein is cholic acid.

14. The nanoparticles according to any one of claims 11 to 13, wherein the conjugates of formula (II) are crosslinked via thiol-containing crosslinkable groups.

15. The nanoparticles according to any one of claims 11 to 14, wherein the conjugates of formula (I) and formula (II) are in a ratio of about 1:1 (w / w).

16. The nanoparticles of claim 15, wherein the nanoparticles have an average diameter of about 20 nm.

17. The nanoparticle of any one of claims 10 to 16, further comprising a therapeutic agent in the hydrophobic core.

18. The therapeutic agents include gardiquimod, doxorubicin, daunorubicin, vincristine, vinblastine, paclitaxel, SN-38, imiquimod, resiquimod, motlimod, lenalidomide, pomalidomide, and the compounds of the structure (S2): 【Chemistry 5】 18. The nanoparticle of claim 17, wherein the nanoparticle is selected from the group consisting of LLS30 having the formula:

19. A pharmaceutical composition for treating a disease, comprising a therapeutically effective amount of nanoparticles according to any one of claims 10 to 18.

20. 20. The pharmaceutical composition of claim 19, wherein the disease is cancer.

21. 21. The pharmaceutical composition of claim 19 or 20, wherein the disease is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma or metastatic cancer.

22. 22. The pharmaceutical composition of any one of claims 19 to 21, further comprising a therapeutic agent in said hydrophobic core, said therapeutic agent being selected from the group consisting of doxorubicin, gardiquimod and imiquimod.

23. 23. The pharmaceutical composition of claim 22, wherein the therapeutic agent is imiquimod, for use in combination with a checkpoint blockade antibody.

24. The pharmaceutical composition of claim 23, wherein the checkpoint blockade antibody is anti-PD-1, anti-PDL1, or anti-CTLA-4.

25. The pharmaceutical composition of claim 23 or 24, wherein the checkpoint blocking antibody is anti-PD-1.

26. 20. A pharmaceutical composition for treating a disease via photothermal therapy in a subject in need thereof, comprising a therapeutically effective amount of the nanoparticles of any one of claims 10 to 18, used prior to exposing the subject to radiation, thereby treating the disease via photothermal therapy.

27. 27. The pharmaceutical composition of claim 26, wherein the subject is exposed to near-infrared light irradiation.

28. 28. The pharmaceutical composition of claim 26 or 27, wherein the disease is cancer.

29. The pharmaceutical composition according to any one of claims 26 to 28, wherein the disease is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma or metastatic cancer.

30. 30. The pharmaceutical composition of any one of claims 26 to 29, further comprising a therapeutic agent in said hydrophobic core, said therapeutic agent being selected from the group consisting of doxorubicin, gardiquimod and imiquimod.

31. The pharmaceutical composition of claim 30, wherein the therapeutic agent is imiquimod, for use in combination with administration of anti-PD-1 via injection.

32. A pharmaceutical composition for use in a method for treating a subject suffering from diseased tissue, said pharmaceutical composition comprising an effective amount of the nanoparticles of any one of claims 10 to 18, said method comprising: administering the pharmaceutical composition to a subject such that the nanoparticles concentrate in the diseased tissue; illuminating the diseased tissue with a first wavelength to identify the diseased tissue; and removing said diseased tissue from said subject, thereby treating said subject; 13. A pharmaceutical composition comprising:

33. 33. The pharmaceutical composition of claim 32, wherein the method further comprises delaying the irradiating step for at least 3 hours after the administering step.

34. 34. The pharmaceutical composition of claim 32 or 33, wherein the method further comprises irradiating any remaining diseased tissue in the subject with a second wavelength.

35. 35. The pharmaceutical composition of claim 34, wherein the ablation and both irradiation steps are performed during a single operation on the object.

36. 35. The pharmaceutical composition of claim 34, wherein the first wavelength and the second wavelength are in the near infrared spectrum.

37. The pharmaceutical composition according to any one of claims 32 to 36, wherein the diseased tissue is cancer.

38. The pharmaceutical composition according to any one of claims 32 to 37, wherein the diseased tissue is breast cancer, oral cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, brain cancer, melanoma, myeloma, leukemia, colon cancer, prostate cancer, esophageal cancer, liver cancer, head and neck cancer, lymphoma, or metastatic cancer.

39. The pharmaceutical composition according to any one of claims 32 to 38, wherein the diseased tissue is ovarian cancer.

40. 1. A composition for use in an imaging method, comprising: A composition comprising an effective amount of the nanoparticles according to any one of claims 10 to 18.

41. A method for preparing nanoparticles according to any one of claims 10 to 18, comprising the steps of:

10. A method comprising forming a reaction mixture comprising a plurality of first conjugates and an aqueous solution, each of the plurality of first conjugates being a compound according to any one of claims 1 to 9, and wherein the conjugates self-assemble to form a nanoparticle, such that a hydrophobic core is formed in the interior of the nanoparticle, and wherein the PEG of each conjugate self-assembles on the exterior of the nanoparticle.

42. The reaction mixture comprises a compound of formula (II): 【Chemistry 6】 wherein PEG is a polyethylene glycol (PEG) polymer, said PEG polymer having a molecular weight of 1-100 kDa; each X is a branched monomer unit; each Y is a thiol-containing crosslinkable group; each L is a linking group, and Each R 1 and are independently cholic acid or a cholic acid derivative.

43. 43. The method of claim 41 or 42, wherein the aqueous solution comprises PBS, and the method further comprises sonicating the reaction mixture for about 10 minutes.

44. 44. The method of claim 42 or 43, wherein the conjugates of formula (I) and formula (II) are added in a ratio of about 1:1 (w / w).

45. 45. The method of any one of claims 42 to 44, wherein in formula (II), each Y is a cysteine ​​and the crosslink is formed via oxidation of the cysteine.

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