Sequential targeting of crosslinked nanotherapeutics for the treatment of brain tumors

Abstract

The present invention provides a compound of formula (I) as defined herein. The present invention also provides nanoparticles comprising a plurality of the conjugates of the present invention, and methods of using the nanoparticles for drug delivery, treating diseases, and imaging.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 949,284, filed on December 17, 2019, the entire content of which is incorporated herein by reference for all purposes.

[0003] Statement of rights to inventions made under federally - sponsored research and development

[0004] This invention was made with government support under Grant No. R01CA199668 awarded by the National Institutes of Health / National Cancer Institute and Grant No. R01HD086195 awarded by the National Institutes of Health / Eunice Kennedy Shriver National Institute of Child Health and Human Development. The government has certain rights in this invention. Background of the Invention

[0005] Multiple drug delivery barriers severely impede the efficacy of brain tumor treatment, including severe blood circulation instability effects, the blood - brain barrier / blood - brain tumor barrier (BBB / BBTB), and limited tumor uptake. The present invention relates to a sequential targeting cross - linking (STICK) nanodelivery strategy to circumvent these important physiological barriers to improve drug delivery to brain tumors. STICK nanoparticles (STICK - NPs) can sequentially target the BBB / BBTB and brain tumor cells with surface maltobionic acid (MA) and 4 - carboxyphenylboronic acid (CBA), respectively, while enhancing the stability of the nanoparticles through pH - responsive cross - linking formed in situ by MA and CBA. STICK - NPs exhibit a longer circulation time (17 - fold higher area under the curve) than free reagents, thus increasing the chance of crossing the BBB / BBTB through glucose transporter - mediated transcytosis by MA. The tumor acidic environment then triggers the transformation of STICK - NPs into smaller nanoparticles, enabling the secondary CBA targeting group for deep tumor penetration and enhancing tumor cell uptake. In mice with invasive and chemoresistant diffuse intrinsic pontine glioma, STICK - NPs significantly inhibit tumor growth and prolong survival with limited toxicity. The formulation addresses multiple physiological barriers on - demand through a simple and intelligent STICK design. Thus, these properties enable STICK - NPs to unlock the potential of brain tumor therapies to improve their therapeutic efficacy.

[0006] Patients with invasive brain tumors, such as glioblastoma multiforme (GBM) or diffuse intrinsic pontine glioma (DIPG) in children, have a poor prognosis. Especially for DIPG, a destructive and invasive pediatric brain tumor that occurs in the ventral pons, radiotherapy is currently the only treatment option. The five-year survival rate of children with DIPG is only about 2%. Many chemotherapeutic drugs, such as vincristine (VCR), and novel epigenetic regulators, such as histone deacetylase (HDAC) inhibitors, bromodomain and extra-terminal motif (BET) bromodomain inhibitors, and enhancer of zeste homolog 2 (EZH2) have shown promising results in preclinical models. Unfortunately, compared with radiotherapy alone, all clinical trials of chemotherapy and epigenetic regulators have failed to improve the treatment outcome. The clinical therapeutic effect of these drugs is significantly hindered by poor drug delivery to brain tumors caused by several physiological barriers, including strong unstable conditions during blood circulation (barrier 1), blood-brain barrier (BBB) / blood-brain tumor barrier (BBTB) (barrier 2), poor specificity for targeting tumor cells (barrier 3), and the relatively weak enhanced permeability and retention effect exhibited by brain tumors ( Figure 1 a). There is an urgent need to develop new treatment strategies for brain tumors.

[0007] It has been reported that various nanocarriers have attempted to bypass these biological barriers by actively targeting receptors or transporters on the BBB / BBTB (such as glucose transporter 1 (GLUT1), transferrin receptor, low-density lipoprotein receptor, choline transporter, and amino acid transporter) and tumor cells / tissues (such as sialic acid, integrin family, tropomyosin receptor kinase (TRK) family proteins, epidermal growth factor receptor (EGFR), and folate receptor), respectively. The BBB / BBTB is a highly regulated barrier that controls the entry of blood-borne substances into the parenchyma of the central nervous system (CNS) and prevents the entry of toxic substances, including chemotherapeutic drugs. Several nutrients, including glucose, are essential for the brain. GLUT1 facilitates the transport of glucose into the CNS, and GLUT1 is specifically localized to the BBB / BBTB. Several studies have identified GLUT1 as an effective target for transporter-mediated transcytosis of nanoparticles. It is also known that many types of tumor cells (including brain tumor cells) exhibit increased sialic acid expression on membrane glycoproteins. Excessive sialylation of the cell membrane during malignant transformation not only contributes to tumor growth and metastasis but is also closely associated with poor prognosis in cancer patients. Therefore, targeting tumor cells through abnormal sialylation has become an attractive strategy for cancer treatment. GLUT1 and sialic acid are targeted by different nanocarriers, but dual / sequential targeting has never been achieved with a single particle design.

[0008] To address the challenges of brain tumor delivery, the design of multifunctional nanoparticles must take into account the entire process of drug delivery to brain tumors and the kinetic requirements at each delivery stage. Several dual-targeting strategies have been developed to attempt to overcome the multiple barriers in brain tumor delivery. For example, the dual-targeting polypeptide angiopep-2 was conjugated to nanoparticles to target the BBB and GBM cells, and this dual-targeting nanocarrier was shown to have excellent anti-intracranial GBM effects. Polysorbate 80 (PS80) was introduced into polymer-conjugated trastuzumab (anti-Her2 antibody) to target the BBB and Her2+ breast cancer brain metastases. In this system, the first step involves PS80-mediated recruitment of circulating apolipoproteins leading to transcytosis, and the second step is the targeting of Her2 on breast cancer cells with trastuzumab after nanoparticle dissociation. Although conceptually appealing, these traditional dual-targeting designs are typically achieved by simply conjugating one or two different targeting moieties on the nanoparticle surface. These moieties are only used for targeting purposes and do not add various favorable physical characteristics to the nanoparticle platform to ingeniously address the complex issues in brain tumor delivery.

[0009] The present invention developed a simple and effective sequential targeting crosslinking (STICK) nanodelivery method to improve drug delivery to brain tumors. Strategically, a unique pair of targeting molecules, maltobionic acid (MA, a glucose derivative) and 4-carboxyphenylboronic acid (CBA), were selected as dual-targeting moieties for the BBB and brain tumors via GLUT1 and sialic acid, respectively, to construct interlocked STICK nanoparticles (STICK NPs). In addition to the targeting function, this pair of targeting moieties can form pH-sensitive borate bonds to stabilize the nanocarrier via intermicellar crosslinking, thus facilitating the stability of NPs in blood circulation ( Figure 1 a, Barrier 1). GLUT1 can recognize the excess MA (a glucose derivative) on the nanoparticle surface and then trigger GLUT1-mediated BBB / BBTB transcytosis ( Figure 1 a, Barrier 2). After exposure to the acidic extracellular pH in solid tumors, the intrinsic MA-CBA borate crosslinking bond cleaves, resulting in the transformation of STICK NPs into small secondary nanoparticles with a new uncovered surface of CBA (a synthetic lectin mimic), which allows for deeper tumor penetration and recognition of tumor surface sialic acid, respectively ( Figure 1 a, Barrier 3). In this study, a stepwise demonstration of the kinetic properties was provided, especially the design to overcome each barrier using the STICK method, including their sequential targeting ability, pharmacokinetics, and pH-dependent drug release / transformation characteristics. Finally, their excellent anticancer targeting ability was demonstrated using dual-modal imaging and anticancer efficacy in two different invasive orthotopic brain tumor models. Summary of the Invention

[0010] In one embodiment, the present invention provides a compound of formula I: (R 1 ) m -D 1 -L 1 -PEG-L 2 -D 2 -(R 2 ) n (I), wherein: each R 1 is independently a peptide, a 1,2-dihydroxy compound or a boronic acid derivative; each R 2 is independently a cholic acid or a cholic acid derivative; D 1 and D 2 are each independently a dendrimer having a single central group and a plurality of branched monomer units X; each branched monomer unit X is a diamino carboxylic acid, a dihydroxy carboxylic acid or a hydroxy amino carboxylic acid; L 1 and L 2 are each independently a chemical bond or a linker sequence connecting to the central group of the dendrimer; PEG is a polyethylene glycol (PEG) polymer having a molecular weight of 1-100 kDa; the subscript m is an integer from 2 to 8; the subscript n is an integer between 2 and 16.

[0011] In another embodiment, the present invention provides a nanoparticle comprising a plurality of first and second conjugates, wherein: each first conjugate is a compound of formula I, wherein each R 1 is independently a peptide, a 1,2-dihydroxy compound, a sugar compound glucose or a glucose derivative; each second conjugate is a compound of formula I, wherein each R 1 is independently a boronic acid derivative; and the plurality of conjugates self-assemble by forming crosslinking bonds to form a nanoparticle such that the interior of the nanoparticle contains a hydrophilic interior that contains a plurality of micelles having a hydrophobic core.

[0012] In another embodiment, the present invention provides a nanoparticle comprising a hydrophilic exterior and an interior, wherein the interior of the nanoparticle contains a hydrophilic interior that contains a plurality of micelles having a hydrophobic core and a hydrophilic micelle exterior, wherein each micelle contains a plurality of first and second conjugates, wherein: each first conjugate is a compound of formula I, wherein each R 1 is independently a peptide, a 1,2-dihydroxy compound, a sugar compound glucose or a glucose derivative; each second conjugate is a compound of formula I, wherein each R 1 is independently a boronic acid derivative; the plurality of first and second conjugates self-assemble by forming crosslinking bonds to form a micelle having a hydrophobic core, wherein the crosslinking bonds are located on the hydrophilic micelle exterior.

[0013] In another embodiment, the present invention provides a method for delivering a drug, the method comprising: administering the nanoparticles of the present invention, wherein the nanoparticles further comprise a hydrophilic and / or hydrophobic drug and a plurality of crosslinking bonds; and in-situ cleavage of the crosslinking bonds such that the drug is released from the nanoparticles, thereby delivering the drug to a subject in need thereof.

[0014] In another embodiment, the present invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of the nanoparticles of the present invention to a subject in need thereof, wherein the nanoparticles further comprise a hydrophilic and / or hydrophobic drug.

[0015] In another embodiment, the present invention provides an imaging method, comprising: administering an effective amount of the nanoparticles of the present invention to a subject in need thereof, wherein the nanoparticles further comprise a hydrophilic and / or hydrophobic contrast agent; and imaging the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 a shows the design of convertible STICK-NPs and a detailed multi-barrier resolution mechanism for brain tumors. The targeting group pair selected to form sequential targeted crosslinking (STICK) is maltobionic acid (MA) (a glucose derivative), and carboxylphenylboronic acid (CBA) (a boronic acid), and is constructed into a well-characterized self-assembled micelle formulation (PEG-CA8). STICK-NPs are assembled from a pair of MA4-PEG-CA8 and CBA4-PEG-CA8 with a molar ratio of 9:1. When intermicellar borate crosslinking occurs, MA and CBA form STICK, resulting in a larger nanoparticle size. An excess of MA groups is located on the surface of the nanoparticles, while CBA groups are first covered within the STICK to avoid non-specific binding. The hydrophobic drug is loaded in the hydrophobic core of the secondary small micelles, while the hydrophile is trapped in the hydrophilic space between the small micelles. In the following studies, a variety of control micelle formulations were used, including NM (non-targeted), MA-NPs (single BBB-targeted), and CBA-NPs (single sialic acid tumor-targeted) nanoparticles (see the inserted table). Specifically, STICK-NPs can overcome barrier 1 (unstable conditions in the blood) through an intermicellar crosslinking strategy, overcome barrier 2 (BBB / BBTB) through active GLUT1-mediated transcytosis across brain endothelial cells, and overcome barrier 3 (penetration and tumor cell endocytosis) by converting into secondary smaller micelles and displaying a secondary active targeting group (CBA) to counteract overexpressed sialic acid on tumor cells, thus responding to the acidic extracellular pH in solid tumors. Figure 1 b shows the intensity-weighted distributions of MA-NPs, CBA-NPs, NM, and STICK-NPs at pH 7.4 and 6.5. Figure 1c shows the formation of borate ester bonds verified by fluorescence assay based on alizarin red S (ARS) indicator (Ex: 468 nm, 0.1 mg / mL). The ARS fluorescence decreased in a dose-dependent manner with the increase of the concentration of MA4-PEG-CA8 from 0 μM to 40 μM (fixing CBA4-PEG-CA8 at 2.5 μM). This demonstrated the formation of borate ester bonds between MA4-PEG-CA8 and CBA4-PEG-CA8. Figure 1 d shows transmission electron micrograph (TEM) imaging to observe the process of transformation of STICK-NPs (92 ± 21 nm) into secondary small micelles (14 ± 3 nm) when changing from pH 7.4 to pH 6.5 at 10 minutes (intermediate state) and 24 hours. The sizes of the large micelles and secondary small micelles measured by TEM were more consistent with those measured by DLS (at pH 7.4: 113.6 ± 45.4 nm and at pH 6.5: 14 ± 3 nm respectively) in the number-weighted distribution ( Figure 8 f). It should be noted that the low-contrast nanoparticle profile in the intermediate state represents empty large nanoparticles with associated secondary small micelles on the outside. Scale bar, 200 nm or 100 nm (insert). Figure 1 e represents the pH-dependent change in the intensity-weighted distribution of STICK-NPs. Figure 1 f shows the time-dependent change in the intensity-weighted distribution of STICK-NPs at pH 6.5. pH 6.8 seems to be the critical value triggering micelle transformation. Figure 1 g is the Z-average size of STICK-NP formulated in PBS with different solvents (various polarities) and treated or untreated with sodium dodecyl sulfate (SDS). ACN: acetonitrile; DCM: dichloromethane; EtOAc: ethyl acetate.

[0017] Figure 2 a and 2b show the cumulative release curves of hydrophilic (Gd-DTPA)( Figure 2 a) and hydrophobic (Cy7.5) payloads( Figure 2 b) from STICK-NPs and NMs. A mixture of NM and free Gd( Figure 2 a) was used because Gd could not be loaded into NM. The drug release study was initially carried out in PBS at pH 7.4 (gray area), and then after 4 hours, under the condition of pH 6.5 (pink area). Samples were collected at different time points, and the Gd-DTPA level was measured by inductively coupled plasma mass spectrometry (ICP-MS), and the Cy7.5 concentration was measured by fluorescence spectrometer. (n = 3). Figure 2c shows the in vitro T1-weighted MRI signals of different concentrations of Gd-DTPA and STICK-NP@Cy@Gd at pH 7.4 or pH 6.5 obtained by a Bruker Biospec 7T MRI scanner. Figure 2 d shows the Z-average size stability test of STICK-NP@Cy@Gd in the presence of PBS, 10 mg / mL SDS, or 10% FBS. (n = 3). Figure 2 e shows the change in the intensity-weighted distribution of STICK-NPs in the presence of different concentrations of glucose (mmol / L). Notably, the normal human serum glucose level ranges from 3.9 to 5.5 mmol / L. Figure 2 f shows the pharmacokinetic curves of free Cy7.5, STICK-NP@Cy, and NM@Cy (Cy7.5, 10 mg / kg) in jugular vein-catheterized rats (n = 3). Serum was collected at different time points, and the drug concentration was measured based on the fluorescence signal. Error bars are standard deviation (SD).

[0018] Figure 3 a-3m shows the study on the multi-barrier resolution mechanism for STICK-NPs-mediated in vitro brain tumor drug delivery process. Figure 3 a shows barrier 2 (BBB / BBTB), and a schematic diagram of the membrane filter ([[]] 0.4 μm pore size) of STICK-NP@Cy-mediated transcytosis through brain endothelial cells. Mouse brain endothelial cells (bEnd.3) were cultured in the upper chamber. Figure 3 b shows the quantitative measurement of the intracellular fluorescence intensity of Cy7.5 in bEnd.3 cells. bEnd.3 cells were cultured with free Cy7.5, STICK-NP@Cy, MA-NP@Cy, CBA-NP@Cy, and NM@Cy (Cy7.5: 0.1 mg / mL) and lysed at different time points. To inhibit GLUT1 activity, cells were pretreated with 40 μM WZB-117 for 1 hour before the following intracellular endocytosis study ([[]] Figure 3 b-3c). (n = 3, ** p < 0.01, two-way ANOVA). Figure 3 c shows the efficiency of transcytosis of different formulations with Cy7.5 in a membrane filter (Transwell) system (as [[[]] Figure 3 a). Mouse bEnd.3 cells were seeded in the upper chamber to form tight junctions, which were measured by > 200 Ω.cm 2The transendothelial electrical resistance (TEER) was confirmed. Free Cy7.5, MA-NP@Cy, CBA-NP@Cy, NM@Cy, and STICK-NP@Cy were loaded into the upper chamber, and the lower chamber medium was collected at different time points to measure the fluorescence intensity of Cy7.5. Figure 3 d shows the intensity-weighted distributions of CK-NP@Cy in the upper chamber and the lower chamber with the medium adjusted to pH 7.4 and 6.5, respectively. The size was measured by DLS. n = 3. Figure 3 e shows a representative confocal image of the subcellular distribution of STICK-NP@DiD (red) in bEnd.3 cells after 1 hour of culture. Lysosome tracer (Lysotracker, green): lysosome; Hoechst 33342 (blue): nuclear staining; scale bar = 20 μm. Figure 3 f shows the VCR concentration in the normal brain tissue of Balb / c mice with an intact BBB 6 hours after intravenous injection of STICK-NPs@VCR and other formulations (2 mg / kg). The whole brain was homogenized. VCR was extracted and the concentration was measured by liquid chromatography-mass spectrometry (LC-MS). Figure 3 g shows a graph depicting barrier 3 - tumor uptake and pH-dependent transformation, where the newly revealed CBA is used for sialic acid-mediated tumor targeting. Figure 3 h represents the fluorescence quantitative determination of total intracellular Cy7.5 under the same treatment conditions at different time points. The fluorescence intensity of Cy7.5 was measured in lysed cells. n = 3, ** p < 0.01, two-way ANOVA. Scale bar = 20 μm. Figure 3 i and Figure 3 j are the representative quantitative analysis and fluorescence images of the endocytosis of free Cy7.5, MA-NP@Cy, CBA-NP@Cy, NM@Cy, and STICK-NP@Cy (Cy7.5: 0.1 mg / mL) in U87-MG cells at the 1-hour time point under different pH (7.4 and 6.5) conditions. In one parallel group treated with STICK-NPs, the sialic acid expression on the tumor cell surface was increased by 40 μM of azidothymidine (AZT). In another parallel group treated with STICK-NPs, 40 μM of free CBA was added to compete with the surface CBA (secondary targeting group) on the secondary STICK-NPs. n = 3, ** p < 0.01, two-way ANOVA. Figure 3 k is a schematic diagram of a membrane filter (Transwell, 0.4 μm pore size) co-culture system, where bEND3 cells are in the upper chamber and U87-MG cells are in the lower chamber to simulate barrier 2 and 3. Figure 3 l and Figure 3m are representative fluorescence images and quantitative analysis graphs of U87-MG cells after being treated with free Cy7.5, MA-NP@Cy, CBA-NP@Cy, NM@Cy, and STICK-NP@Cy (Cy7.5: 0.1 mg / mL) for 1 hour in the upper chamber. One hour after adding to the upper chamber, the medium in the lower chamber was adjusted to pH 7.4 or 6.5 and maintained for another hour, and the U87-MG cells in the lower chamber were cultured for another hour. In the parallel group treated with STICK-NPs, the activity of GLUT1 was inhibited in advance using WZB-117. Scale bar = 20 μm. Error bars are standard deviation (SD).

[0019] Figure 4 a - 4d show the transformation-dependent tumor penetration study of STICK-NPs. Figure 4 a is the quantitative analysis of the penetration of STICK-NP@DiD (pH 7.4 and 6.5) and other formulations (pH 7.4) in U87-MG-GFP neurospheres. The Z-average size of STICK-NP@DiD (pH 7.4) is approximately 155 nm, while that of STICK-NP@DiD (pH 6.5) and other nanopreparations is approximately 20 nm. n = 3. t-test, ** P < 0.01. Figure 4 b are the representative images and quantitative analysis graphs of the penetration of STICK-NP@DiD (red) into DIPG tumor spheres at 24 hours under the conditions of pH 7.4 and 6.5. (DiD: 0.05 mg / mL). n = 3. t-test, ** P < 0.01. Scale bar, 100 μm. Figure 4 c shows the tissue penetration of STICK-NP@DiD in the normal brain region and the DIPG-implanted region in an in situ mouse model 16 hours after injecting STICK-NP@DiD and NM@DiD (red, 5 mg / kg). An in situ model was established by injecting DIPG-XIII-P cells into the mouse brainstem. STICK-NP@DiD and NM@DiD (red, 5 mg / kg) were injected into the mice bearing DIPG for 16 hours. Before sacrificing the mice, fluorescein isothiocyanate-labeled dextran (Dextran-FITC) (green, molecular weight = 70K) was injected to label blood vessels. ImageJ (right) was used to analyze the penetration distance of blood vessels. DAPI (blue): nuclear staining. Scale bar = 100 μm. Figure 4 d is the tissue penetration analysis of STICK@DiD and NM@DiD (red) outside blood vessels (FITC, green) in the normal brain and DIPG tumor sites corresponding to Figure 4 the cross-section (yellow line) in c.

[0020] Figure 5a-5f is the delivery process of dual-modal imaging (MRI and NIRF imaging)-guided STICK-NPs in in situ human-derived tissue xenograft (PDX) glioblastoma and PDX DIPG brain tumor models. Figure 5 a shows in vivo T1-weighted MRI and NIRF images (in vivo and ex vivo) at specified time points after intravenous injection of Cy7.5+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, NM@Cy+Gd, or STICK-NP@Cy@Gd (Gd-DTPA: 25 mg / kg; Cy7.5: 10 mg / kg) into glioblastoma PDX mouse models. Since hydrophilic Gd-DTPA cannot be loaded onto MA-NP, CBA-NP, or NM, free Gd-DTPA combined with Cy7.5-loaded nanoparticles was used as a control. Tumor location was double-verified by T2-weighted MR imaging. Figure 5 b is the quantitative analysis of MRI T1 signal intensity normalized to normal brain tissue. t-test, ** p < 0.01. Figure 5 c is the NIRF intensity analysis of in situ brain tumors based on whole-mouse in vivo imaging at 24 and 48 hours after injection. n = 3, t-test, ** p < 0.01, * p < 0.05. Figure 5 d is a biodistribution analysis plot of mice bearing PDX GBM based on Cy7.5 fluorescence intensity (ex vivo NIRF imaging) at 24 hours after injection of Cy7.5+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, NM@Cy+Gd, and STICK-NP@Cy@Gd. n = 3, t-test, ** p < 0.01. Figure 5 e are representative confocal images of cryosections of the brains of mice implanted with GBM tumors at 24 hours after injection of Cy7.5+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, NM@Cy+Gd, and STICK-NP@Cy@Gd. Blue: DAPI; Green: U87-MG-GFP; Red: Cy7.5. Scale bar = 500 μm. Error bars are standard deviation (SD). Figure 5 f are T1-weighted MRI images and confocal fluorescence images with quantitative analysis of the in situ PDX DIPG brain tumor model at 24 hours after administration of NM@Cy+Gd or STICK-NP@DiD@Gd (Gd-DTPA: 25 mg / kg; DiD: 5 mg / kg). Before sacrificing the mice, animals were injected with fluorescein isothiocyanate-labeled dextran (Dextran-FITC, green) to label blood vessels. Red: DiD; Scale bar = 2 mm.

[0021] Figure 6 a-6e shows the anti-cancer efficacy study of STICK-NPs@VCR in an orthotopic PDX DIPG mouse model. Figure 6 a shows the tumor progression (blue dashed outline) of an orthotopic DIPG mouse model monitored by Gd-enhanced T1-weighted MRI on days 0, 6, 12, 18, and 24 of the same representative mice in each group after treatment (intravenous injection) every six days with PBS, free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, STICK-NP@VCR, vincristine sulfate liposome (Marqibo) (VCR 1.5 mg / kg) free VCR2, and STICK-NM@VCR2 (VCR 2 mg / kg). Scale bar = 10 mm. Figure 6 b is Figure 6 The actual tumor burden (blue dashed outline) confirmed by histopathology after injection on day 12 of the same representative mice with MRI results in a. Scale bar = 5 mm. Figure 6 c is the quantitative analysis of the MRI-based tumor growth curve, Figure 6 d is the Kaplan-Meier survival curve, Figure 6 e is the graph of body weight changes in mice bearing DIPG after treatment with STICK-NP, vincristine sulfate liposome (Marqibo), and other formulations. n = 6. A t-test was performed for tumor burden analysis; a log-rank (Mantel-Cox) test was performed for survival time analysis. ** p < 0.01, * p < 0.05. Notably, all mice in the PBS, free VCR, NM@VCR, MA-NP@VCR, and CBA-NP@VCR treatment groups died after day 12, while there were survivors in the STICK-NP@VCR group. Therefore, the tumor growth curve and body weight changes after day 12 were plotted only based on the mice that survived in the STICK-NP@VCR group.

[0022] Figure 7 a-7j are the characterization diagrams of CBA4-PEG-CA8 and MA4-PEG-CA8 linear-dendritic block copolymers. Figure 7 a is the synthesis process and chemical structure of CBA4-PEG-CA8 and MA4-PEG-CA8 linear-dendritic block copolymers. Figure 7 b are the MALDI-TOF MS and gel permeation chromatography (GPC) spectra of NH2-PEG5k-NH2 polymer, CBA4-PEG-CA8 terminal dendritic copolymer (telodendrimer), and MA4-PEG-CA8 terminal dendritic copolymer. Figure 7c is the 1H NMR spectrum of CBA4-PEG-CA8 in CDCl 3 3 1 , and Figure 7 d is the 1H NMR spectrum of MA4-PEG-CA8 in CDCl 3 3 1 . The chemical shifts of the PEG chain (3.5 - 3.7 ppm), cholic acid (0.5 - 2.4 ppm), and the attached MA (3.2 - 4.5 ppm) can be observed at the characteristic peaks of the 1H NMR spectrum of MA4-PEG-CA8 in CDCl3. The chemical shifts of the PEG chain (3.5 - 3.7 ppm), cholic acid (0.5 - 2.4 ppm), and the attached CBA (7.2 - 8.4 ppm) can be observed at the characteristic peaks of the 1H NMR spectrum of CBA4-PEG-CA8 in CDCl3. Figure 7 e shows the effect of the ratio of the two end-dendritic copolymers on the size, Figure 7 and Figure 7 f shows the effect of the ratio of the two end-dendritic copolymers on the PdI, (n = 3). Figure 7 g is a representative fluorescence image and quantitative expression map of the endocytosis of the ratio of the two end-dendritic copolymers on brain endothelial cells (bEND.3) by loading DiD dye (red). Hoechst fluorescent dye (blue): nuclear staining. Figure 7 h is the size distribution diagram (number-weighted) of MA-NPs, CBA-NPs, NM, and STICK-NPs at pH 7.4 and 6.5, Figure 7 i is the pH-dependent size change (number-weighted) diagram of STICK-NPs, Figure 7 and j is the time-dependent size change diagram (number-weighted) of STICK-NPs at pH 6.5. pH 6.8 seems to be the critical value for triggering micelle transformation. Error bars are standard deviation (SD).

[0023] Figure 8 a - 8f are the characterizations of STICK-NP@Cy@Gd. Figure 8 a is the TEM image of MA-NPs micelles, Figure 8 b is the TEM image of CBA-NPs micelles. The micelle concentration is maintained at 1.0 mg / mL. Figure 8 c is the fluorescence spectrum of STICK-NP@Cy@Gd (Cy7.5: 0.02 mg / mL) in PBS. Ex / Em = 820 / 848 nm. Figure 8 d is the relaxation rate (r1) of STICK-NP@Cy@Gd at pH 7.4, Figure 8 and Figure 8 e is the relaxation rate of STICK-NP@Cy@Gd at pH 6.5. Figure 8f is the intensity (left panel) and number (right panel) weighted distributions of STICK-NP at pH 7.4 (upper panel) and pH 6.5 (lower panel). Summary table of nanoparticle sizes measured by different methods. The number weighted distribution emphasizes smaller nanoparticles more and is generally more consistent with the findings in TEM or cryo-electron microscopy (Cryo-EM). The slight size difference between the peak average value + / - SD in TEM and the number weighted distribution is because TEM measures the dry size while DLS measures the hydrodynamic size.

[0024] Figure 9 Indicates that WZB-117 (GLUT1 inhibitor, 40 μM) inhibits the surface expression of GLUT1 in brain endothelial cells. Immunofluorescent localization (a) and quantitative expression (b) of GLUT1 in brain endothelial cells (bEND.3) with WZB-117 (positive control: untreated; negative control: without GLUT1 antibody). c) Quantitative analysis of the BBB penetration efficiency after incubating different VCR formulations in the BBB model system of a membrane filter (Transwell, 0.4 μm pore size) for 1 hour with bEND.3 cells seeded in the upper chamber. Error bars are standard deviation (SD).

[0025] Figure 10 Indicates the BBB / BBTB transverse efficiency of STICK-NPs. Uptake of free Cy, MA-NP@Cy, CBA-NP@Cy, NM@Cy, and STICK-NP@Cy by brain endothelial cells (bEND.3) was observed by confocal microscopy and quantitative fluorescence intensity. In another group, bEND.3 cells were pretreated with WZB-117 (GLUT1 inhibitor) and then incubated with STICK-NP@Cy. Scale bar = 40 μm.

[0026] Figure 11 Are representative images of the penetration of STICK-NP@DiD (red) into U87-MG-GFP (green) tumor spheroids at 24 hours under conditions of pH 7.4 and 6.5. (DiD, 0.05 mg / mL). Scale bar = 100 μm. White dotted line: maximum penetration depth.

[0027] Figure 12 a - 12d show dual-modal imaging-guided drug delivery of STICK-NPs in orthotopic GBM (PDX) brain tumor mice. Figure 12 a is in vivo whole-brain MR imaging of orthotopic PDX brain tumor-bearing mice at different time points after injection of Cy+Gd, NM@Cy+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, and STICK-NP@Cy@Gd (Cy7.5: 10 mg / kg, Gd-DTPA: 25 mg / kg). Figure 12b shows in vivo NIR fluorescence imaging of orthotopic PDX brain tumor-bearing mice at different time points after injection with Cy+Gd, NM@Cy+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, and STICK-NP@Cy@Gd (Cy 7.5: 10 mg / kg, Gd-DTPA: 25 mg / kg). Figure 12 c shows ex vivo NIR fluorescence imaging of orthotopic PDX brain tumor-bearing mice at different time points after injection with Cy+Gd, NM@Cy+Gd, MA-NP@Cy+Gd, CBA-NP@Cy+Gd, and STICK-NP@Cy@Gd (Cy 7.5: 10 mg / kg, Gd-DTPA: 25 mg / kg). Ex vivo imaging was at the 24-hour time point. Figure 12 d shows enhanced representative confocal images of cryosections of the brains of mice bearing PDX tumors at 24 hours after injection with STICK-NP@Cy@Gd, focusing on the tumor area. Blue: DAPI; Green: U87-MG-GFP; Red: Cy 7.5. Scale bar = 500 μm.

[0028] Figure 13 Tumor growth data plotted for the PBS, free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, STICK-NP@VCR, vincristine sulfate liposomes (VCR 1.5 mg / kg), free VCR2, and STICK-NP@VCR2 (VCR 2 mg / kg) groups are based on MRI.

[0029] Figure 14 Body weight change data plotted for the PBS, free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, STICK-NP@VCR, vincristine sulfate liposomes (VCR 1.5 mg / kg), free VCR2, and STICK-NP@VCR2 (VCR 2 mg / kg) groups are shown.

[0030] Figure 15 a shows MR imaging monitoring orthotopic U87-MG tumors (red arrows) at days 0, 6, 12, and 18 after treatment with PBS, free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, and STICK-NP@VCR (VCR 2 mg / kg). Scale bar = 10 mm. Figure 15 b shows quantitative analysis of the tumor growth curve based on MRI. n = 4, t-test, ** p < 0.01. Figure 15 c shows the Kaplan–Meier plot of survival of orthotopic U87-MG mice treated. (n = 4). Log-rank (Mantel-Cox) test,* p < 0.05. Figure 15 d is the histopathological evaluation of sections of brain / U87-MG brain tumors (black arrows) on day 12 after injection. Scale bar = 5 mm. Error bars are standard deviation (SD). Figure 15 e is the body weight change of U87-MG orthotopic brain tumor-bearing mice treated with PBS, VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, and STICK-NP@VCR on day 1 and day 12 (VCR: 2 mg / kg). (n = 4). Figure 15 f is the histopathological evaluation of major organs of orthotopic U87-MG brain tumor-bearing mice treated with PBS, VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, and STICK-NP@VCR (VCR: 2 mg / kg) on day 12 after initial treatment (scale bar = 200 μm, hematoxylin and eosin (H&E) staining). Error bars are standard deviation (SD). Detailed implementation mode

[0031] I. General methods

[0032] The present invention provides a terminal dendritic copolymer, wherein one end contains cholic acid or its derivative, and the other end contains a peptide, a 1,2-dihydroxy compound, or a boric acid derivative, which can form a nanocarrier through crosslinking. The nanocarrier contains multiple at least two different conjugates that can be crosslinked and can contain hydrophilic and hydrophobic drugs inside. The nanocarrier can be used for drug delivery, disease treatment, and imaging.

[0033] II. Definitions

[0034] Unless otherwise specifically stated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, any method or material similar or equivalent to the methods or materials described herein can be used to implement the present invention. For the purposes of the present invention, the following terms are defined.

[0035] As used herein, "a", "an", or "the" includes aspects of not only one member but also multiple members. For example, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes multiple such cells, a reference to "a reagent" includes a reference to one or more reagents known to those skilled in the art, and so on.

[0036] "Peptide" refers to a compound containing two or more amino acids covalently linked by peptide bonds. As used herein, the term includes amino acid chains of any length, including full-length proteins.

[0037] "1,2-dihydroxy compound" refers to a compound having at least two hydroxyl groups on adjacent carbon atoms. 1,2-dihydroxy compounds include, but are not limited to, sugars, glucose, glucose derivatives, cellulose, oligosaccharides, cyclodextrins, maltobionic acid, glucosamine, sucrose, trehalose, and cellobiose.

[0038] "Boric acid derivative" refers to a compound having a -B(OH) 2 functional group. Examples of boric acid derivatives include, but are not limited to, 3-carboxy-5-nitrophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 4-(hydroxymethyl)phenylboronic acid, 5-bromo-3-carboxyphenylboronic acid, 2-chloro-4-carboxyphenylboronic acid, 2-chloro-5-carboxyphenylboronic acid, 2-methoxy-5-carboxyphenylboronic acid, 2-carboxy-5-pyridineboronic acid, 6-carboxy-2-fluoropyridine-3-boronic acid, 5-carboxy-2-fluoropyridine-3-boronic acid, 4-carboxy-3-fluorophenylboronic acid, and 4-(bromomethyl)phenylboronic acid.

[0039] "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. 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) cholan-3α,7α,12α-triol; cholic acid; and choline. Cholic acid derivatives and analogs, such as, but not limited to, allocholic acid, chenodeoxycholic acid, hyodeoxycholic acid, deoxycholic acid, and chenodeoxycholic acid, can also be used in the present invention. Cholic acid derivatives can be designed to modulate the properties of nanocarriers generated by the assembly of terminal dendritic copolymers, such as micelle stability and membrane activity. For example, cholic acid derivatives can have a hydrophilic surface modified with one or more glycerol groups, aminopropanediol groups, or other groups.

[0040] "Monomer" and "monomeric unit" refer to diamino carboxylic acids, dihydroxy carboxylic acids or hydroxy amino carboxylic acids. Examples of the diamino carboxylic acid groups of the present invention include, but are not limited to, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropionic acid, 3-amino-2-aminomethyl-2-methylpropionic acid, 4-amino-2-(2-aminoethyl)butyric acid and 5-amino-2-(3-aminopropyl)pentanoic acid. Examples of the 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 hydroxy amino carboxylic acids include, but are not limited to, serine and homoserine. Those skilled in the art will understand that other monomeric units can be used in the present invention.

[0041] "Diamino carboxylic acid" refers to a compound containing two amine functional groups and at least one carboxyl functional group.

[0042] "Dihydroxy carboxylic acid" refers to a compound containing two hydroxyl functional groups and at least one carboxyl functional group.

[0043] "Hydroxy amino carboxylic acid" refers to a compound containing at least one hydroxyl functional group and at least one amine functional group.

[0044] "Nanoparticle" or "nanocarrier" refers to a particle or carrier formed by aggregation of the micelles described in the present invention. The nanoparticle or nanocarrier can be spherical in shape with a diameter ranging from 1 to 500 nanometers or more. The nanocarrier described in the present invention has a hydrophilic interior and a hydrophilic exterior containing micelles.

[0045] "Micelle" refers to an aggregate of the compounds described in the present invention. The micelle described in the present invention has a hydrophobic core and a hydrophilic exterior, which is part of the internal environment of the nanoparticle.

[0046] "Drug" refers to an agent capable of treating and / or ameliorating a disorder or disease. The drug can be a hydrophobic drug, i.e., any drug that repels water, or a hydrophilic drug, which can be dissolved in water. Hydrophobic drugs that can be used in the present invention include, but are not limited to, deoxycholic acid, taxanes, doxorubicin, etoposide, irinotecan, paclitaxel (PTX), docetaxel, epothilones (epothilone classes), rapamycin and platinum drugs. Hydrophilic drugs that can be used in the present invention include, but are not limited to, gemcitabine, doxorubicin hydrochloride (DOX·HCl) and cyclophosphamide. Other drugs include non-steroidal anti-inflammatory drugs and vinca alkaloids such as vinblastine and vincristine. The drugs described in the present invention also include prodrug forms. Those skilled in the art will understand that other drugs can be used in the present invention.

[0047] "Imaging" refers to using a device external to a subject to determine the location of a contrast agent, such as a compound of the present invention. Examples of imaging tools include, but are not limited to, fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), and x-ray computed tomography (CT).

[0048] "Contrast agent" refers to a compound used in an imaging method to increase the contrast of structures within a cell or body location, including but not limited to fluorescence microscopy, MRI, PET, SPECT, and CT. A contrast agent can emit radiation, fluorescence, a magnetic field, or radio waves. Contrast agents include, but are not limited to, radiometal chelates, radiometal atoms or ions, and fluorophores.

[0049] "Administering" refers to administering to a subject orally, as a suppository, by topical contact, parenterally, intravenously, intraperitoneally, intramuscularly, intralesionally, intranasally, or subcutaneously, intrathecally, or by implanting a sustained-release device, such as a micro-osmotic pump.

[0050] A subject refers to an animal such as a mammal, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the subject is a human.

[0051] "Therapeutically effective amount" or "therapeutically sufficient amount" or "effective or sufficient amount" refers to a dose that, when administered, produces a therapeutic effect. The exact dose will depend on the purpose of the treatment and can be determined by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th ed., 2003, Gennaro ed., Lippincott Williams & Wilkins). In sensitized cells, the therapeutically effective dose is typically lower than the conventional therapeutically effective dose for non-sensitized cells.

[0052] "Treat", "treating", or "treatment" means any indication of successful treatment or amelioration of an injury, pathology, disorder, or symptom (such as pain), including any objective or subjective parameter, such as alleviation; remission; making a symptom, injury, pathology, or disorder more tolerable to the patient; reducing the frequency or duration of a symptom or disorder; or, in some cases, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter; including, for example, the results of a physical examination.

[0053] "Disease" means an abnormal condition that negatively affects the structure or function of part or all of an organism, not due to trauma. A disease is generally interpreted as a medical condition associated with specific symptoms and signs. Diseases may include cancer, immunodeficiency, hypersensitivity, allergy, and autoimmune diseases.

[0054] III. Compounds

[0055] In some embodiments, the present invention provides a compound of formula I: (R 1 ) m -D 1 -L 1 -PEG-L 2 -D 2 -(R 2 ) n (I), wherein: each R 1 is independently a peptide, a 1,2-dihydroxy compound, or a boronic acid derivative; each R 2 is independently a bile acid or a bile acid derivative; D 1 and D 2 are each independently a dendrimer having a single central group and a plurality of branched monomer units X; each branched monomer unit X is a diamino carboxylic acid, a dihydroxy carboxylic acid, or a hydroxyamino carboxylic acid; L 1 and L 2 are each independently a chemical bond or a linker sequence connected to the central group of the dendrimer; PEG is a polyethylene glycol (PEG) polymer having a molecular weight of 1-100 kDa; the subscript m is an integer from 2 to 8; the subscript n is an integer between 2 and 16.

[0056] Each R 1 of the present invention may include any suitable peptide, 1,2-dihydroxy compound, or boronic acid derivative known to those skilled in the art.

[0057] In some embodiments, each R 1is a peptide. In some embodiments, the peptide is an oligopeptide, cyclic peptide, dipeptide, tripeptide or tetrapeptide. In some embodiments, the peptide is an oligopeptide, such as angiopep-2, lixisenatide, plecanatide, parsabiv, teriparatide or abaloparatide. In some embodiments, the peptide is angiopep-2.

[0058] In some embodiments, each R 1 is a 1,2-dihydroxy compound. In some embodiments, the 1,2-dihydroxy compound is levodopa, dopamine, cellulose, oligosaccharide, cyclodextrin, maltobionic acid, glucosamine, allose, glucose, mannose, galactose, fructose, sucrose, trehalose or cellobiose. In some embodiments, the 1,2-dihydroxy compound is levodopa, cellulose, oligosaccharide, cyclodextrin, maltobionic acid, glucosamine, sucrose, trehalose or cellobiose. In some embodiments, the 1,2-dihydroxy compound is maltobionic acid.

[0059] In some embodiments, each R 1 is independently a peptide, 1,2-dihydroxy compound, sugar compound, glucose or glucose derivative. In some embodiments, each R 1 is independently angiopep-2, levodopa, cellulose, oligosaccharide, cyclodextrin, maltobionic acid, glucosamine, sucrose, trehalose or cellobiose. In some embodiments, each R 1 is independently maltobionic acid.

[0060] In some embodiments, each R 1 is independently a boronic acid derivative. In some embodiments, the boronic acid derivative is phenylboronic acid, 2-thienylboronic acid, methylboronic acid, cis-propenylboronic acid, trans-propenylboronic acid, 3-carboxy-5-nitrophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 4-(hydroxymethyl)phenylboronic acid, 5-bromo-3-carboxyphenylboronic acid, 2-chloro-4-carboxyphenylboronic acid, 2-chloro-5-carboxyphenylboronic acid, 2-methoxy-5-carboxyphenylboronic acid, 2-carboxy-5-pyridineboronic acid, 6-carboxy-2-fluoropyridine-3-boronic acid, 5-carboxy-2-fluoropyridine-3-boronic acid, 4-carboxy-3-fluorophenylboronic acid or 4-(bromomethyl)phenylboronic acid.

[0061] In some embodiments, each R 1Independently 3-carboxy-5-nitrophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 4-(hydroxymethyl)phenylboronic acid, 5-bromo-3-carboxyphenylboronic acid, 2-chloro-4-carboxyphenylboronic acid, 2-chloro-5-carboxyphenylboronic acid, 2-methoxy-5-carboxyphenylboronic acid, 2-carboxy-5-pyridylboronic acid, 6-carboxy-2-fluoropyridine-3-boronic acid, 5-carboxy-2-fluoropyridine-3-boronic acid, 4-carboxy-3-fluorophenylboronic acid or 4-(bromomethyl)phenylboronic acid. In some embodiments, each R 1 Independently 4-carboxyphenylboronic acid.

[0062] R 2 can be any suitable cholic acid or cholic acid derivative known to those skilled in the art. Cholic acid derivatives and analogs include, but are not limited to, allocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, and chenodeoxycholic acid. Cholic acid derivatives can be designed to modulate the properties of the nanocarriers generated by the assembly of the terminal dendritic copolymers, such as micelle stability and membrane activity. For example, the cholic acid derivative can have a hydrophilic surface modified with one or more glycerol groups, aminopropanediol groups, or other groups.

[0063] In some embodiments, each R 2 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-bis(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-NH 2 ). In some embodiments, each R 2 is cholic acid.

[0064] In some embodiments, each branched-chain monomer unit X can be a diamino carboxylic acid, a dihydroxy carboxylic acid, or a hydroxyamino carboxylic acid. In some embodiments, X is a diamino carboxylic acid. In some embodiments, each diamino carboxylic acid can be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropionic acid, 3-amino-2-aminomethyl-2-methylpropionic acid, 4-amino-2-(2-aminoethyl)butyric acid, or 5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, each dihydroxy carboxylic 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 hydroxyamino carboxylic acid can be serine or homoserine. In some embodiments, the diamino carboxylic acid is an amino acid.

[0065] In some embodiments, each X is independently 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine, 3-amino-2-aminomethylpropionic acid, 3-amino-2-aminomethyl-2-methylpropionic acid, 4-amino-2-(2-aminoethyl)butyric acid, and 5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, each X is lysine.

[0066] L of the present invention 1 is a chemical bond or any suitable linker sequence. In some embodiments, L 1 is a chemical bond. In some embodiments, L 1 is a linker sequence. The linker sequence can be any suitable linker sequence known to those skilled in the art. In some embodiments, the linker sequence is C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, a PEG polymer, or a peptide. In some embodiments, the linker sequence is C 1-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, or a PEG polymer.

[0067] L of the present invention 2 is a chemical bond or any suitable linker sequence. In some embodiments, L 2 is a chemical bond. In some embodiments, L 2 is a linker sequence. The linker sequence can be any suitable linker sequence known to those skilled in the art. In some embodiments, the linker sequence is C 1-20 alkylene, C 2-20 alkenylene, C2-20 An alkynylene, a PEG polymer, or a peptide. In some embodiments, the linker sequence is C 1-10 An alkylene, C 2-10 An alkenylene, C 2-10 An alkynylene, or a PEG polymer.

[0068] Polyethylene glycol (PEG) polymers of any size and structure can be used in the present invention. In some embodiments, the PEG has a molecular weight of 1 - 100 kDa. In some embodiments, the PEG has a molecular weight of 1 - 50 kDa. In some embodiments, the PEG has a molecular weight of 1 - 20 kDa. In some embodiments, the PEG has a molecular weight of 1 - 10 kDa. In some embodiments, the PEG has a molecular weight of 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 has a molecular weight of about 5 kDa. Those skilled in the art will understand that other PEG polymers and other hydrophilic polymers can be used in the present invention. The PEG can be of any suitable length.

[0069] The subscript m and the subscript n can be any suitable integers. In some embodiments, the subscript m is an integer from 2 to 8. In some embodiments, the subscript m is an integer from 3 to 6. In some embodiments, the subscript m is 4. In some embodiments, the subscript n is an integer from 2 to 16. In some embodiments, the subscript n is an integer from 4 to 12. In some embodiments, the subscript n is an integer from 6 to 10. In some embodiments, the subscript n is 8. In some embodiments, the subscript m is 4 and the subscript n is 8.

[0070] In some embodiments, the compound has the structure shown in formula (Ia):

[0071]

[0072] In some embodiments, the compound has the structure shown in formula (Ib):

[0073]

[0074] In some embodiments, the present invention provides a compound shown in formula (Ib), wherein: each R 1 is maltobionic acid; each R 2 is cholic acid; each X is lysine; the molecular weight of the PEG is about 5 kDa.

[0075] In some embodiments, the present invention provides a compound shown in formula (Ib), wherein each R 1 is 4 - carboxyphenylboronic acid; each R 2is cholic acid; each X is lysine; the molecular weight of PEG is about 5 kDa.

[0076] IV. Nanoparticles

[0077] In some embodiments, the present invention provides nanoparticles comprising a plurality of first conjugates and second conjugates, wherein: each first conjugate is a compound of formula I, wherein each R 1 is independently a peptide, a 1,2-dihydroxy compound, a saccharide compound glucose or a glucose derivative; each second conjugate is a compound of formula I, wherein each R 1 is independently a boronic acid derivative; and the plurality of conjugates self-assemble by forming cross-linking bonds to form nanoparticles such that the interior of the nanoparticles contains a hydrophilic interior that contains a plurality of micelles having a hydrophobic core.

[0078] In some embodiments, the present invention provides nanoparticles comprising a hydrophilic exterior and an interior, wherein the interior of the nanoparticles contains a hydrophilic interior that contains a plurality of micelles having a hydrophobic core and a hydrophilic micelle exterior, wherein each micelle contains a plurality of first conjugates and second conjugates, wherein: each first conjugate is a compound of formula I, wherein each R 1 is independently a peptide, a 1,2-dihydroxy compound, a saccharide compound glucose or a glucose derivative; each second conjugate is a compound of formula I, wherein each R 1 is independently a boronic acid derivative; the plurality of first conjugates and second conjugates self-assemble by forming cross-linking bonds to form micelles having a hydrophobic core, wherein the cross-linking bonds are located on the hydrophilic micelle exterior.

[0079] The first conjugate and the second conjugate can be any suitable compound of the present invention. In some embodiments, the first conjugate and the second conjugate are independently compounds of formula (Ia). In some embodiments, the first conjugate and the second conjugate are independently compounds of formula (Ia) or formula (Ib). In some embodiments, the first conjugate is a compound of formula (Ib), wherein R 1 is a peptide, a 1,2-dihydroxy compound, a saccharide compound, glucose or a glucose derivative. In some embodiments, the first conjugate is a compound of formula (Ib), wherein R 1 is an angiopep-2, levodopa, cellulose, oligosaccharide, cyclodextrin, maltobionic acid, glucosamine, sucrose, trehalose or cellobiose. In some embodiments, the first conjugate is a compound of formula (Ib), wherein R 1 is maltobionic acid.

[0080] In some embodiments, the second conjugate is a compound of formula (Ib), wherein R 1is a boric acid derivative. In some embodiments, the second conjugate is a compound of formula (Ib), wherein, R 1 is 3-carboxy-5-nitrophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 4-(hydroxymethyl)phenylboronic acid, 5-bromo-3-carboxyphenylboronic acid, 2-chloro-4-carboxyphenylboronic acid, 2-chloro-5-carboxyphenylboronic acid, 2-methoxy-5-carboxyphenylboronic acid, 2-carboxy-5-pyridylboronic acid, 6-carboxy-2-fluoropyridine-3-boronic acid, 5-carboxy-2-fluoropyridine-3-boronic acid, 4-carboxy-3-fluorophenylboronic acid or 4-(bromomethyl)phenylboronic acid. In some embodiments, the first conjugate is a compound of formula (Ib), wherein, R 1 is 4-carboxyphenylboronic acid.

[0081] In some embodiments, the first conjugate is a compound of formula (Ib), wherein: each R 1 is maltaric acid; each R 2 is cholic acid; each X is lysine; PEG has a molecular weight of about 5 kDa, and the second conjugate is a compound of formula (Ib), wherein, each R 1 is 4-carboxyphenylboronic acid; each R 2 is cholic acid; each X is lysine; PEG has a molecular weight of about 5 kDa.

[0082] In some embodiments, the nanoparticles further comprise a hydrophilic drug or a contrast agent. In some embodiments, the hydrophilic drug or contrast agent is encapsulated inside the hydrophilic nanocarrier and outside the hydrophilic micelle.

[0083] The hydrophilic drug useful in the present invention can be any suitable hydrophilic drug. In some embodiments, the hydrophilic drug is atenolol, penicillin, ampicillin, lisinopril, vancomycin, cisplatin, gemcitabine, doxorubicin hydrochloride (DOX·HCl) and cyclophosphamide. In some embodiments, the hydrophilic drug is vancomycin, cisplatin, gemcitabine, doxorubicin hydrochloride (DOX·HCl) and cyclophosphamide. In some embodiments, the hydrophilic drug is cisplatin, gemcitabine, doxorubicin hydrochloride (DOX·HCl) and cyclophosphamide.

[0084] The hydrophilic contrast agent useful in the present invention can be any suitable hydrophilic contrast agent. In some embodiments, the hydrophilic contrast agent is calcein, Alexa 680, gadopentetic acid (Gd-DTPA) or indocyanine green (ICG). In some embodiments, the hydrophilic contrast agent is calcein, gadopentetic acid (Gd-DTPA) or indocyanine green (ICG). In some embodiments, the hydrophilic contrast agent is gadopentetic acid (Gd-DTPA) or indocyanine green (ICG).

[0085] In some embodiments, the hydrophilic drug or contrast agent is gadopentetic acid (Gd-DTPA), indocyanine green (ICG), cisplatin, gemcitabine, doxorubicin hydrochloride (DOX·HCl), or cyclophosphamide.

[0086] In some embodiments, the nanoparticles further comprise a hydrophobic drug or contrast agent. In some embodiments, the hydrophobic drug or contrast agent is encapsulated in the hydrophobic core of the micelles inside the nanoparticles.

[0087] The hydrophobic drugs useful in the present invention can be any suitable hydrophobic drugs. In some embodiments, the hydrophobic drug is resiquimod, gardiquimod, imiquimod, doxorubicin (DOX), vincristine (VCR), everolimus, carmustine, lomustine, temozolomide, lenvatinib mesylate, sorafenib tosylate, regorafenib, irinotecan, paclitaxel (PTX), docetaxel, BET inhibitor, OTX015, BET-d246, ABBV-075, I-BET151, I-BET 762, HDAC inhibitor, valproic acid, vorinostat, panobinostat, entinostat, ricolinostat, AR-42, JMJD3 inhibitor, GSKJ4, EZH2 inhibitor, tazemetostat, GSK2816126, MC3629, EGFR inhibitor, gefitinib, erlotinib, lapatinib, osimertinib, AZD92291, IDH inhibitor, enasidenib, ivosidemib, Notch inhibitor, RO4929097, CDK4 / 6 inhibitor, palbociclib, ribociclib, abemaciclib, PI3K / Akt / mTOR inhibitor, rapamycin, buparlisib, curcumin or etoposide. In some embodiments, the hydrophobic drug is doxorubicin (DOX), vincristine (VCR), everolimus, carmustine, lomustine, temozolomide, lenvatinib mesylate, sorafenib tosylate, regorafenib, irinotecan, paclitaxel (PTX), docetaxel, BET inhibitor, OTX015, BET-d246, ABBV-075, I-BET151, I-BET 762, HDAC inhibitor, valproic acid, vorinostat, panobinostat, entinostat, ricolinostat, AR-42, JMJD3 inhibitor, GSKJ4, EZH2 inhibitor, tazemetostat, GSK2816126, MC3629, EGFR inhibitor, gefitinib, erlotinib, lapatinib, osimertinib, AZD92291, IDH inhibitor, enasidenib, ivosidemib, Notch inhibitor, RO4929097, CDK4 / 6 inhibitor, palbociclib, ribociclib, abemaciclib, PI3K / Akt / mTOR inhibitor, rapamycin, buparlisib, curcumin or etoposide.

[0088] The hydrophobic contrast agents useful in the present invention can be any suitable hydrophobic contrast agents. In some embodiments, the hydrophobic contrast agent is cyanine 5.5 (Cy5.5), cyanine 7.5 (Cy7.5), or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-4-chlorobenzenesulfonate (DiD). In some embodiments, the hydrophobic contrast agent is cyanine 7.5 (Cy7.5) or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-4-chlorobenzenesulfonate (DiD).

[0089] In some embodiments, the hydrophobic drug or contrast agent is cyanine 7.5 (Cy7.5), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-4-chlorobenzenesulfonate (DiD), doxorubicin (DOX), vincristine (VCR), everolimus, carmustine, lomustine, temozolomide, lenvatinib mesylate, sorafenib tosylate, regorafenib, irinotecan, paclitaxel (PTX), docetaxel, BET inhibitor, OTX015, BET-d246, ABBV-075, I-BET151, I-BET762, HDAC inhibitor, valproic acid, vorinostat, panobinostat, entinostat, ricolinostat, AR-42, JMJD3 inhibitor, GSKJ4, EZH2 inhibitor, tazemetostat, GSK2816126, MC3629, EGFR inhibitor, gefitinib, erlotinib, lapatinib, osimertinib, AZD92291, IDH inhibitor, enasidenib, ivosidenib, Notch inhibitor, RO4929097, CDK4 / 6 inhibitor, palbociclib, ribociclib, abemaciclib, PI3K / Akt / mTOR inhibitor, rapamycin, buparlisib, curcumin, or etoposide.

[0090] The ratio of the first conjugate to the second conjugate can be any suitable ratio known to those skilled in the art and is reported as a molar ratio. In some embodiments, the ratio of the first conjugate to the second conjugate is from about 100:1 to 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is from about 50:1 to 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is from about 25:1 to 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is from about 10:1 to 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is about 50:1, 25:1, 10:1, 9:1, 5:1, 1:1, 1:5, or 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is about 10:1, 9:1, 5:1, 1:1, 1:5, or 1:10. In some embodiments, the ratio of the first conjugate to the second conjugate is about 10:1, 9:1, and 5:1. In some embodiments, the ratio of the first conjugate to the second conjugate is about 9:1.

[0091] V. Pharmaceutical Composition Formulations

[0092] The compositions of the present invention can be formulated into a variety of oral, parenteral, and topical dosage forms. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, elixirs, suspensions, etc., suitable for ingestion by a patient. The compositions of the present invention can also be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, duodenally, or intraperitoneally. In addition, the compositions described herein can be administered by inhalation, e.g., intranasally. In addition, 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, insufflations, powders, and aerosol formulations (e.g., steroid inhalers, see Rohatagi, Journal of Clinical Pharmacology. 35:1187-1193, 1995; Tjwa, Annals of Allergy, Asthma & Immunology. 75:107-111, 1995). Accordingly, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of the present invention.

[0093] For the preparation of pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Preparations in solid form include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier can be one or more substances which may also function as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials. Details of formulation and administration techniques are described in scientific and patent literature, see, for example, the latest edition of "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, Pennsylvania ("Remington's").

[0094] In powders, the carrier is a finely divided solid which is mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain from 5% or 10% to 70% of the compound of the present invention.

[0095] 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; and gums, including gum arabic and tragacanth; and proteins, including but not limited to gelatin and collagen. If desired, disintegrating agents or solubilizing agents can be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts, such as sodium alginate.

[0096] The cores of dragees have suitable coatings, such as concentrated sugar solutions which may also contain gum arabic, talc, polyvinylpyrrolidone, carbomer gels, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablet or dragee coatings for product identification or to characterize the amount (i.e., dosage) of the active compound. The pharmaceutical preparations of the present invention can also be used orally, for example, push-fit capsules made of gelatin, and soft-sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the compound of the present invention mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the compound of the present invention can be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol, with or without a stabilizer.

[0097] For the preparation of suppositories, first melt a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, and then uniformly disperse the compound of the present invention therein by stirring. Then pour the molten homogeneous mixture into a suitably sized mold and allow it to cool so as to solidify.

[0098] Formulations in liquid form include solutions, suspensions and emulsions, such as aqueous or water / propylene glycol solutions. For parenteral injection, the liquid formulation can be formulated in a solution in an aqueous polyethylene glycol solution.

[0099] Aqueous solutions suitable for oral use can be prepared by dissolving the compound of the present invention in water and adding suitable colorants, flavorants, stabilizers and thickeners as required. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water containing a viscous substance such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia, and a dispersing or wetting agent such as a naturally occurring phospholipid (such as lecithin), a condensation product of an alkylene oxide and a fatty acid (such as polyoxyethylene stearate), a condensation product of ethylene oxide and a long-chain fatty alcohol (such as heptadecaethyleneoxycetanol), a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol (such as polyoxyethylene sorbitan monooleate), or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride (such as polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or propyl p-hydroxybenzoate, one or more colorants, one or more flavorants and one or more sweeteners such as sucrose, aspartame or saccharin. The formulation can be adjusted according to the osmotic pressure.

[0100] Also included are solid-form formulations which are intended to be converted into liquid-form formulations shortly before use for oral administration. Such liquid forms include solutions, suspensions and emulsions. In addition to the active ingredient, these formulations may also contain colorants, flavorants, stabilizers, buffering agents, artificial and natural sweeteners, dispersing agents, thickeners, solubilizers and the like.

[0101] The oil suspension can be formulated by suspending the compounds of the present invention in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil or a mineral oil such as liquid paraffin; or a mixture of these. The oil suspension can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. A sweetening agent can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These preparations can be preserved by adding an antioxidant such as ascorbic acid. For an example of an injectable oil carrier, see Minto, Journal of Pharmacological and Experimental Therapeutics (J.Pharmacal.Exp.Ther.). 281:93-102, 1997. The pharmaceutical preparations of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil or a mineral oil, as described above, or a mixture of these. Suitable emulsifying agents include naturally occurring gums, such as gum arabic and tragacanth, naturally occurring phospholipids, 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. The emulsion can also contain a sweetening agent and a flavoring agent, such as syrups and elixirs in the formulation. Such preparations can also contain demulcents, preservatives or coloring agents.

[0102] The compositions of the present invention can also be delivered as microspheres for slow release in vivo. For example, the microspheres can be formulated for administration by intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, Journal of Biomaterials Science - Polymer Edition (J.Biomater Sci.Polym.). 7th Ed.: 623-645, 1995); as biodegradable and injectable gel formulations (see, e.g., Gao, Pharmaceutical Research (Pharm.Res). 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, Journal of Pharmacy and Pharmacology. 49:669-674, 1997). Both transdermal and intradermal routes can provide sustained delivery for weeks or months.

[0103] In another embodiment, the compositions of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a body cavity or lumen of an organ. Formulations for administration generally comprise a solution of the composition of the present invention dissolved in a pharmaceutically acceptable carrier. Acceptable carriers and solvents that can be used include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile, fixed oils are commonly used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic mono- or di-glycerides of fatty acids. In addition, fatty acids such as oleic acid can also be used in the preparation of injectables. These solutions are sterile and generally free of undesirable substances. These formulations can be sterilized by conventional, well-known sterilization techniques. As required, the formulations can contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the composition of the present invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., and according to the particular mode of administration selected and the needs of the patient. For intravenous administration, the formulation can be a sterile injectable formulation, such as a sterile injectable aqueous or oleaginous suspension. Such suspensions can be formulated using those suitable dispersing or wetting agents and suspending agents in accordance with the prior art. The sterile injectable formulation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution.

[0104] In another embodiment, the formulations of the compositions of the present invention can be delivered by liposomes that fuse with cell membranes or are endocytosed, i.e., by using ligands that are linked to liposomes or directly to the oligonucleotide and that bind to cell surface membrane protein receptors, resulting in endocytosis. By using liposomes, particularly when ligands specific for the target cells are carried on the liposome surface or otherwise targeted preferentially to a particular organ, the compositions of the present invention can be delivered in vivo to target cells in a concentrated manner. (See, e.g., Al-Muhammed, J Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J Hasp. Pharm. 6:1576-1587, 1989).

[0105] VI. Administration

[0106] The compositions of the present invention can be delivered by any suitable means, including oral, parenteral, and topical methods. Transdermal administration methods by the topical route can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0107] The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses each containing a suitable quantity of the compound of the invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, such as packaged tablets, capsules and powders in vials or ampoules. Additionally, the unit dosage form can be a capsule, tablet, cachet or lozenge by itself, or can be any of these dosage forms in a suitable number of packaged forms.

[0108] The compound of the invention can be present in any suitable amount and can depend on various factors including, but not limited to, the weight and age of the subject, the disease state, etc. Suitable dosage ranges of the compound of the invention include from about 0.1 mg to about 10,000 mg, or from about 1 mg to about 1000 mg, or from about 10 mg to about 750 mg, or from about 25 mg to about 500 mg, or from about 50 mg to about 250 mg. Suitable dosages of the compound of the invention include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.

[0109] The compound of the invention can be administered at any suitable frequency, interval and duration. For example, the compound of the invention can be administered once per hour, or two, three or more times per hour, once per day, or two, three or more times per day, or once every 2, 3, 4, 5, 6 or 7 days to provide a preferred dosage level. When the compound of the invention is 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 compound of the invention can be administered once, twice or three or more times for 1 hour, 1 to 6 hours, 1 to 12 hours, 1 to 24 hours, 6 to 12 hours, 12 to 24 hours, one day, 1 to 7 days, one week, 1 to 4 weeks, one month, 1 to 12 months, one year or longer, even indefinitely.

[0110] The composition can also contain other compatible therapeutic agents. The compounds described herein can be used in combination with another, with other active agents known to be useful for modulating glucocorticoid receptors or with adjuvants that may be ineffective by themselves but may contribute to the efficacy of the active agent.

[0111] The compounds 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 simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequentially in any order the compound of the present invention and the active agent. In addition, the compounds and active agents of the present invention can each be administered once a day, or two, three or more times a day to provide a preferred daily dose level.

[0112] In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising the compound of the present invention and the active agent. In other embodiments, the compound of the present invention and the active agent can be formulated separately.

[0113] The compound of the present invention and the active agent can be present in the compositions of the present invention in any suitable weight ratio, e.g., from 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 compound of the present invention and other active agents can be present in any suitable weight ratio, e.g., 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 dosages and dosage ratios of the compound of the present invention and the active agent are applicable to the compositions and methods of the present invention.

[0114] VII. Methods of Treatment

[0115] In some embodiments, the present invention provides a method of delivering a drug, the method comprising: administering a nanoparticle of the present invention, wherein the nanoparticle further comprises a hydrophilic and / or hydrophobic drug and a plurality of crosslinking bonds; and in-situ cleaving the crosslinking bonds such that the drug is released from the nanoparticle, thereby delivering the drug to a subject in need thereof.

[0116] The nanoparticles of the present invention can comprise a plurality of crosslinking bonds that can be cleaved in-situ under suitable pH conditions such that the drug is released from the nanoparticles. In some embodiments, the pH is 7 or less. In some embodiments, the pH is about 6.5 or less. In some embodiments, the pH is from 1 to 7. In some embodiments, the pH is from 1 to 6.5. In some embodiments, the pH is from 2 to 6.5. In some embodiments, the pH is from 4 to 6.5. In some embodiments, the pH is about 4, 4.5, 5, 5.5, 6 or 6.5. In some embodiments, the pH is about 6.5.

[0117] The hydrophobic drugs that can be used in the present invention can be any hydrophobic drugs known to those skilled in the art. The hydrophobic drugs that can be used in the present invention include but are not limited to deoxycholic acid, deoxycholates, resiquimod, gardiquimod, imiquimod, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, taxusin A, taxusin B, or taxusin C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, epothilones (epothilone class), rapamycin, and platinum drugs. The hydrophilic drugs that can be used in the present invention include but are not limited to atenolol, penicillin, ampicillin, lisinopril, vancomycin, cisplatin, gemcitabine, doxorubicin hydrochloride (DOX·HCl), and cyclophosphamide. Other drugs include non-steroidal anti-inflammatory drugs and vinca alkaloids such as vinblastine and vincristine.

[0118] The drugs that can be used in the present invention include chemotherapeutic agents and immunomodulators. For example, the drug can be but is not limited to deoxycholic acid or deoxycholates in salt form, pembrolizumab, nivolumab, cemiplimab, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, taxusin A, taxusin B, or taxusin C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, epothilones (epothilone class), rapamycin, and platinum drugs. Other drugs include non-steroidal anti-inflammatory drugs and vinca alkaloids such as vinblastine and vincristine. In some embodiments, the drug is paclitaxel, resiquimod, gardiquimod, or deoxycholate.

[0119] In some embodiments, the hydrophilic and / or hydrophobic drug is doxorubicin hydrochloride (DOX·HCl), doxorubicin (DOX), vincristine (VCR), or paclitaxel (PTX).

[0120] In some embodiments, the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the nanoparticles of the present invention, wherein the nanoparticles further comprise a hydrophilic and / or hydrophobic drug.

[0121] The nanocarriers of the present invention can be administered to a subject for treating diseases, including cancer, such as but not limited to: carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer and Burkitt 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, bone cancer, skin cancer, retinoblastoma, multiple myeloma, Hodgkin lymphoma and non-Hodgkin lymphoma (for other cancers, see CANCER: PRINCIPLES AND PRACTICE, DeVita, V.T. et al., 2008 edition).

[0122] 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, drug allergy, insect bite allergy; inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, ileitis and enteritis; vaginitis; psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis; spondyloarthropathy; scleroderma; respiratory allergic diseases, such as asthma, allergic rhinitis, allergic lung diseases, etc., (2) autoimmune diseases, such as arthritis (rheumatoid and psoriatic), osteoarthritis, multiple sclerosis, systemic lupus erythematosus, diabetes, glomerulonephritis, etc., (3) graft rejection (including allograft rejection and graft-versus-host disease), and (4) other diseases that require suppression of unwanted inflammatory responses (e.g., atherosclerosis, myositis, neurological disorders 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).

[0123] In some embodiments, the disease is cancer. In some embodiments, the disease is selected from the group consisting of: bladder cancer, brain cancer, brain metastases, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, diffuse pontine glioma, bowel cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer and uterine cancer. In some embodiments, the disease is selected from the group consisting of: bladder cancer, breast cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, leukemia, lung cancer, myeloma, ovarian cancer and pancreatic cancer.

[0124] In some embodiments, the disease is cancer. In some embodiments, the disease is glioblastoma, diffuse pontine glioma, brain metastases, lung cancer, breast cancer, colon cancer, kidney cancer, or melanoma.

[0125] Hydrophilic and hydrophobic drugs useful in the present invention are as listed above. In some embodiments, the hydrophilic and / or hydrophobic drug is doxorubicin hydrochloride (DOX·HCl), doxorubicin (DOX), vincristine (VCR), or paclitaxel (PTX).

[0126] VI II. Imaging methods

[0127] In some embodiments, the present invention provides an imaging method, comprising: administering to a subject in need an effective amount of the nanoparticles of the present invention, wherein the nanoparticles further comprise a hydrophilic and / or hydrophobic contrast agent; and imaging the subject.

[0128] Imaging techniques useful in the present invention are any suitable techniques known to those skilled in the art. In some embodiments, the imaging technique is positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), X-ray computed tomography (CT), echocardiogram, fluorescence spectroscopy, near-infrared fluorescence (NIRF) spectroscopy, or a combination thereof. In some embodiments, the imaging technique is MRI, fluorescence spectroscopy, NIRF spectroscopy, or a combination thereof. In some embodiments, the imaging technique is MRI, NIRF spectroscopy, or a combination thereof.

[0129] Contrast agents useful in the present invention can be any contrast agents known to those skilled in the art. The contrast agents of the present invention can be hydrophobic or hydrophilic contrast agents. Contrast agents include, but are not limited to, paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are contrast agents that have magnetism under an applied 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 at one radiation wavelength and detection at a second different radiation wavelength. Optical probes useful in the present invention include, but are not limited to, indocyanine green (ICG), Cy5.5, Cy7.5, 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.

[0130] In some embodiments, the hydrophilic and / or hydrophobic contrast agent is gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA), indocyanine green (ICG), cyanine 7.5 (Cy7.5), or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD).

[0131] IX. Examples

[0132] Example 1. Synthesis of the Terminal Dendritic Copolymer

[0133] Chemical reagents: O-(2-aminoethyl)-O'-[2-(tert-butoxycarbonyl-amino)ethyl] decaethylene glycol (NH2-PEG-Boc, molecular weight: 5000 Da) and O-(2-aminoethyl) polyethylene glycol (NH2-PEG, molecular weight: 5000 Da) were purchased from Rapp Polymere (Germany). 4-Carboxyphenylboronic acid (CBA) and maltobionic acid (MA) were purchased from Combi-Blocks Inc. (San Diego, California). (Fmoc)lys(Boc)-OH was purchased from AnaSpec Inc. (San Jose, California). Gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) was purchased from Alizarin Red S (ARS), and cyclohexanone, phosphorus oxychloride (POCl3), 1,1,2-trimethylbenzene-1H-benzo[e]indole, 3-iodopropionic acid, sodium dodecyl sulfate (SDS), D-fructose, cholic acid, azidothymidine (AZT), and all other chemical reagents were purchased from Sigma-Aldrich (St. Louis). The CY7.5 dye was synthesized in the laboratory.

[0134] Synthesis of PEG-CA8, Boc-NH-PEG-CA8, MA4-PEG-CA8 and CBA4-PEG-CA8 terminal dendritic copolymers. PEG5k-CA8 terminal dendritic copolymer and Boc-NH-PEG-CA8 terminal dendritic copolymer were synthesized from NH2-PEG and NH2-PEG-Boc respectively according to the previously reported methods to prepare non-crosslinked micelles (NM) and precursors for crosslinked micelles. MA4-PEG-CA8 and CBA4-PEG-CA8 terminal dendritic copolymers were synthesized by solution-phase condensation reaction using Boc-NH-PEG-CA8 as the starting material as described above. Briefly, the Boc group on Boc-NH-PEG-CA8 was removed by treatment with 50% (v / v) trifluoroacetic acid in dimethylformamide (DMF), and NH2-PEG-CA8 was precipitated by adding cold diethyl ether and then washed twice with cold diethyl ether. (Fmoc)Lys(Fmoc)-OH (4eq.) was coupled to the N-terminus of NH2-PEG-CA8 using DIC and HOBt as coupling reagents until a negative Kaiser test result was obtained, indicating the completion of the coupling reaction to form (Fmoc)Lys(Fmoc)-PEG-CA8. Then the polymer was precipitated by adding cold diethyl ether and washed twice with cold diethyl ether. Then, the polymer was treated with 20% (v / v) 4-methylpiperidine in dimethylformamide (DMF) to remove the Fmoc group, followed by the precipitation and washing steps as described above. The white powder precipitate was dried in vacuo and subjected to two couplings of (Fmoc)Lys(Fmoc)-OH to produce the second-generation dendritic polylysine capped with four Fmoc groups at one end of PEG-CA8. MA and CBA were coupled to the termini of the dendritic polylysine after Fmoc removal to produce MA4-PEG-CA8 terminal dendritic copolymer and CBA4-PEG-CA8 terminal dendritic copolymer respectively. Then the two terminal dendritic copolymers were dialyzed and finally lyophilized.

[0135] The mass spectra of the terminal dendritic copolymers were collected on an ABI 4700 MALDI-TOF / TOF mass spectrometer (linear mode) using 2,5-dihydroxybenzoic acid as the matrix. The molecular weight distribution and polydispersity index (PdI) were collected by gel permeation chromatography (GPC, Waters e2695, mobile phase 0.1M aqueous NH4Ac). The 1H-NMR spectra of the polymers were recorded on a Bruker 800MHz Avance nuclear magnetic resonance spectrometer using CDCl 3 as the solvent.

[0136] Example 2. Nanoparticles

[0137] Preparation of nanoparticles: MA4-PEG-CA8 and CBA4-PEG-CA8 (in different ratios) were first dissolved in certain polar solvents, such as chloroform, in a round-bottom flask. The solvent was evaporated under vacuum to form a thin film. PBS buffer was added to rehydrate the thin film, followed by sonication for 30 minutes. Borate bonds were formed between CBA and MA of adjacent terminal dendrimers, and cross-linked STICK-NPs were formed after self-assembly in PBS. The nanoparticle solution was filtered through a 0.22 μm filter to sterilize the sample. Similarly, in 1 mL of PBS, NM micelles, MA-NPs micelles, and CBA-NPs micelles were prepared by using 10 mg of PEG-CA8, 9 mg of MA4-PEG-CA8 and 1 mg of PEG-CA8, 1 mg of CBA4-PEG-CA8 and 9 mg of PEG-CA8, respectively. No cross-linking was formed in these three control micelles.

[0138] Characterization of nanoparticles: The size and size distribution of the nanoparticles were measured by a dynamic light scattering (DLS) instrument (Malvern, Nano-ZS). For DLS measurement, the concentration of the terminal dendrimer of the nanoparticles was maintained at 1.0 mg / mL. At room temperature, each sample was measured 3 times at the sampling time. The data were analyzed by Malvern Zetasizer software, and the data values were reported as the average of each three measurements. The morphology of the nanoparticles was observed on a TALOS L120C transmission electron microscope (TEM) at pH 7.4 and 6.5 (at the 10th minute and 24 hours). An aqueous solution of the nanoparticles (1.0 mg / mL) was deposited on a copper grid and measured at room temperature. The 1H-NMR spectrum of the terminal dendrimer was recorded using a Bruker 800 MHz spectrometer in CDCl 3 3.

[0139] Study on the formation of STICK-NPs: MA4-PEG-CA8 (0.9 mg) and CBA4-PEG-CA8 (0.1 mg) were dissolved in 1 mL of water, methanol, acetonitrile (ACN), dichloromethane (DCM), ethyl acetate, and toluene, respectively, and the sizes of these nanoparticles were detected by DLS. Then, the solvent was evaporated under vacuum to form a thin film. PBS buffer (1 mL) was added to rehydrate the thin film, followed by sonication for 30 minutes. The sizes and morphologies of these nanoparticles were detected by DLS and TEM. In addition, 0.1 mL of a 20 mg / mL SDS solution was added to these nanoparticles to detect the formation of borate cross-linking by DLS.

[0140] Table 1. Loading rates of STICK-NPs (20 mg / mL) for hydrophilic and hydrophobic agents.

[0141]

[0142] The principle of the STICK method is to select two different targeting groups that can also form stimulus-responsive crosslinks. Considering Barriers 2 and 3 in brain tumor delivery, MA, a glucose derivative, is selected for GLUT1-mediated endocytic transport across BBB / BBTB endothelial cells, and CBA is a boronic acid that can target sialic acid highly expressed on brain tumor cells. A pair of terminal dendritic copolymers, MA4-PEG-CA8 and CBA4-PEG-CA8, (as Figure 1 a; Figure 7 a), the molecular weights, polydispersity indices (PdI), and chemical structures of the two terminal dendritic copolymers are characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), gel permeation chromatography (GPC) (as Figure 7 b) and 1H nuclear magnetic resonance spectroscopy (1H-NMR) (as Figure 7 c-7d). Similar to PEG-CA8, the MA4-PEG-CA8 and CBA4-PEG-CA8 terminal dendritic copolymers can individually form distinct small (Z-average size: ~24 nm) spherical nanoparticles with a narrow size distribution (as Figure 1 b, Figure 7 e-7f and Figure 8 a-8b). To achieve sequential targeting, for the first stage of brain endothelial cells, a higher proportion of MA terminal dendritic copolymer is required so that after forming borate ester bonds with a lower proportion of CBA terminal dendritic copolymer (as Figure 1 c), free MA targeting groups can be left on the nanoparticle surface. Therefore, different ratios (1:1, 5:1, and 9:1) of MA4-PEG-CA8 and CBA4-PEG-CA8 are mixed to form STICK-NPs. The intensity-weighted distribution, polydispersity index (PdI), and brain endothelial cell targeting ability are evaluated using dynamic light scattering (DLS) and fluorescence images respectively (as Figure 7 e-7g). It is found that as the proportion of MA4-PEG-CA8 increases, the size and endothelial cell targeting ability of the resulting nanoparticles increase, and the nanoparticle PdI decreases. Considering all the above factors, the 9:1 ratio of MA4-PEG-CA8 and CBA4-PEG-CA8 is determined to be the optimal ratio because this formulation gives the most uniform nanoparticles (lowest PdI) among all ratios. Other ratios seem to form both large and small nanoparticles simultaneously, indicating possible increased intra-micelle crosslinking (formed within small micelles). Different from the small micelles formed based on one terminal dendritic copolymer (observed by TEM to be approximately 14 nm) (as Figure 8(a - 8b), STICK - NPs are relatively large (Z - average size: 144 nm; TEM size: 92 ± 21 nm), spherical, and contain many smaller secondary micelles, the size of which is comparable to that of non - crosslinked micelles (as Figure 1 b, 1d). As the pH value decreases (from 7.4 to 6.5), the borate ester bonds degrade, and STICK - NPs decompose into many smaller secondary micelles (Z - average size: ∼25 nm, as Figure 1 B; TEM size: 14 ± 3 nm, as Figure 1 d). It should be noted that the Z - average size and intensity - weighted distribution are only used in this study to better describe the transformation process. However, the number - weighted distribution of STICK - NPs at pH 7.4 and 6.5 is also included in Figure 8 f to better explain the findings from TEM detection (as Figure 1 d). The critical pH value for the pH - dependent transformation of STICK - NPs is approximately 6.8 (as Figure 1 e), and after exposure to an environment with pH 6.5, the transformation occurs as early as 5 minutes and is completed in about 1 hour (as Figure 1 f).

[0143] Another special property of STICK - NPs is their ability to encapsulate both hydrophobic and hydrophilic payloads, which has a significant advantage compared to traditional micelles that usually only load hydrophobic drugs. As reported in another study, STICK - NPs selectively self - assemble into core - reversible micelles driven by hydrophilic interactions in low - polarity solvents and form a large amount of hydrophilic space. The formation of inter - micellar cross - links preserves the hydrophilic space together with the newly formed hydrophobic core during the subsequent aqueous solution assembly process. This enables the capture of hydrophiles between the hydrophobes in the secondary micelles and the hydrophobic cholate core, just like other control micelles (as Figure 1 a). It has been demonstrated that both hydrophiles (such as indocyanine green (ICG), gadopentetic acid (Gd - DTPA), doxorubicin hydrochloride (DOX·HCl)) and hydrophobes (such as cyanine 7.5 (Cy7.5), 1,1’ - dioctadecyl - 3,3,3’,3’ - tetramethylindodicarbocyanine - 4 - chlorobenzenesulfonate (DiD), vincristine (VCR), and paclitaxel (PTX)) can be encapsulated into STICK - NPs with high loading efficiency (Table 1). Gd - DTPA and Cy7.5 can be co - loaded into STICK - NPs with a diameter of 146 nm for various theranostic applications, as shown in the subsequent section.

[0144] STICK - NPs are formulated in a variety of solvents with different polarities ( Figure 1g). In non-polar solvents, even after solvent evaporation and rehydration in PBS, the size of the reversible micelles remained above 116 nm. Even strong detergents such as sodium dodecyl sulfate (SDS) could not decompose the micelles because MA4-PEG-CA8 and CBA4-PEG-CA8 were able to form stable intermolecular cross-links of micelles in the presence of non-polar solvents. In contrast, in polar solvents, MA4-PEG-CA8 and CBA4-PEG-CA8 could not form core-reversible micelles, and the final nanoparticles showed smaller sizes compared to other control micelles. These smaller micelles were easily disrupted in the presence of SDS (as Figure 1 g), which might be due to the lack of sufficient borate cross-linking bonds to stabilize the nanoparticles.

[0145] Example 3. Drug Delivery

[0146] Loading hydrophobic and hydrophilic agents by STICK-NPs. As described, hydrophobic and hydrophilic agents (Table 1) were loaded into STICK-NPs by the solvent evaporation and cross-linking packaging method. Briefly, hydrophilic agents, MA4-PEG-CA8 (9 mg) and CBA4-PEG-CA8 (1 mg), were dissolved in 2 mL of ultrapure water, then sonicated for 3 minutes, and the water was evaporated under vacuum to form a thin film in a round-bottom flask. Then the thin film and the hydrophobic agent were dispersed in 3 mL of anhydrous chloroform. The chloroform was evaporated under vacuum to form a thin film again. PBS buffer (1 mL) was added to rehydrate the thin film, and then sonicated for 5 minutes. Unloaded free reagents were removed by passing the nanoparticle solution through a centrifugal filtration device (MWCO: 3 kDa, ). STICK-NPs loaded with hydrophobic and hydrophilic agents on the filter were recovered with PBS. The drug loading rate was calculated by absorbance intensity (such as Cy7.5), HPLC (such as vincristine), or inductively coupled plasma mass spectrometry (ICP-MS) (such as Gd-DTPA) according to the calibration curve and standard drug concentration. The loading efficiency was defined as the ratio of the agent loaded into the nanoparticles to the initial agent content.

[0147] Drug release curve: STICK-NP@Cy@Gd was prepared, and a dialysis cassette (Pierce Chemical Company) with a 3 kDa MWCO was used to evaluate the in vitro release curve. To form ideal sink conditions, 10 g of charcoal was added to the release medium. The cassette was dialyzed with PBS (pH 7.4) at room temperature. At 4 hours, PBS with pH 7.4 was replaced with fresh PBS with pH 6.5. The concentrations of CY7.5 and Gd-DTPA retained in the dialysis cassette at different time points were measured by ultraviolet-visible spectrophotometry and ICP-MS.

[0148] Different drug loadings in different compartments of Stick-NPs result in different drug release profiles of hydrophilic and hydrophobic payloads in response to pH changes. Using hydrophilic Gd-DTPA and hydrophobic Cy7.5 dye as model drugs co-loaded into STICK-NPs, drug release studies were initially carried out in a medium with a pH of 7.4, and then, after 4 hours, in a medium with a pH of 6.5 (as Figure 2 a - 2b). This experiment aimed to simulate two stages of in vivo drug release (pH value in blood is 7.4, and pH value in tumor microenvironment is 6.5). Hydrophilic Gd-DTPA cannot be effectively loaded into NMs, so NM + free Gd-DTPA was used in this study. Figure 2 a shows that free Gd-DTPA is released immediately, while Gd-DTPA is released from STICK-NPs at a much lower rate, but the release can be accelerated when the pH is changed to 6.5. This is because hydrophilic Gd-DTPA is trapped between micelles and can diffuse gradually, but can only be released rapidly when the pH-dependent cross-links between micelles are broken. At pH 7.4, the release rate of hydrophobic Cy7.5 loaded into the hydrophobic interior of the secondary micelles of STICK-NPs is significantly lower than that of Gd-DTPA, which may be due to the hydrophobic properties of Cy7.5 (as Figure 2 b). At acidic pH values, the release of Cy7.5 from STICK-NPs is slightly enhanced, which may be due to the formation of slight cross-links within the secondary micelles. In contrast, non-cross-linked non-targeted micelles loaded with Cy7.5 (NM@Cy) show faster drug release at pH 7.4 and minimal response to pH changes because there is no pH-responsive cross-linking (as Figure 2 b). These results indicate that STICK-NP can rapidly release hydrophilic drugs in a low-pH-responsive manner and deliver hydrophobic drugs to tumors through a secondary micelle release mechanism. Utilizing the advantages of co-loading Cy7.5 and Gd-DTPA, STICK-NPs can potentially be applied to dual-modal imaging (magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) imaging) (as Figure 2 c; as Figure 8 c - 8e). After exposure to an environment with a lower pH value, STICK-NP@Cy@Gd is transformed and releases hydrophilic Gd-DTPA, resulting in a restored T1 signal comparable to that of free Gd-DTPA. When the pH changes from 7.4 to 6.5, the r1 of STICK-NP@Cy@Gd increases from 1.061 mM-1*s-1 to 4.447 mM-1*s-1 ( Figure 8 e).

[0149] The first biological barrier to nanoparticle delivery in brain tumors is the strong destabilizing effects in the blood circulation, including extreme dilution, ionic environment, and interactions with blood proteins and lipoproteins (such as HDL, LDL), leading to nanoparticle decomposition and premature drug release. Stabilized by intermolecular crosslinking, STICK-NP@Cy@Gd maintains its size in PBS, and even in the presence of 50 mM SDS and 10% FBS / PBS, STICK-NP@CY@GD maintains its size within 35 days (as Figure 2 d). Since STICK relies on the formation of borate ester bonds between CBA and MA (a glucose derivative with two cis-diols), there is concern that competition from blood glucose may lead to the degradation of the crosslinking. Therefore, additional experiments were conducted and it was demonstrated that the crosslinking is very stable at physiological levels of glucose and up to a glucose concentration of 100 mmol / L (as Figure 2 e). Notably, the blood glucose level in normal individuals is approximately 3.9 - 5.5 mmol / L (70 - 100 mg / dL), and even patients with diabetes are unlikely to reach a blood glucose level of 50 mmol / L. In addition, STICK-NPs showed excellent performance in the pharmacokinetic study of rats. Compared with traditional NMs and free Cy7.5 formulations, the area under the curve (AUC(0-∞)) of STICK-NP@Cy@Gd increased by 5.4-fold and 17.6-fold, respectively (as Figure 2 f; Table 2). In addition, STICK-NP@Cy had the highest Cmax (34.98 ± 3.63 mg / L, or 5 times higher than NM@Cy), and the longest t1 / 2z (34.66 ± 12.13 hours, 2 times longer than NM@Cy). These results strongly demonstrate that STICK-NPs exhibit excellent stability during circulation and prevent premature drug release due to intermolecular crosslinking. This improvement in significantly increasing the systemic circulation time provides an extended drug delivery window for brain tumors.

[0150] Table 2. Pharmacokinetic parameters of various formulations.

[0151]

[0152] Since the in situ brain tumor model may not have an intact BBB due to mechanical damage, it was decided to verify the ability of STICK-NPs to deliver the poorly brain-permeable chemotherapeutic drug VCR in vitro and in normal Balb / c mice. Similarly, compared with free and NM@VCR in the BBB membrane filter (transwell) model system, STICK-NP@VCR can cross brain endothelial cells and deliver significantly higher amounts of VCR to the lower chamber (as Figure 9c). In the Balb / c model, at 6 hours after injection, the whole brain was collected and tissue drug concentration was measured by LC / MS. Compared with free VCR or other non-targeted formulations or single-targeted formulations, after STICK-NP@VCR was confirmed, the amount of VCR retained in normal brain parenchyma was approximately twice (as Figure 3 f). Overall, these results confirmed that STICK-NPs could effectively cross the BBB / BBTB via GLUT1-mediated transcytosis.

[0153] Drug accumulation in brain tissue: Female Balb / c mice (Envigo, Sacramento, CA) at 4 - 5 weeks old were intravenously injected with free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, and STICK-NP@VCR (n = 4) at 2 mg / kg. Six hours later, the animals were killed and the whole brain was obtained immediately. The brain tissue was weighed and homogenized in PBS. VCR was extracted with methanol by 3-minute sonication. Tissue VCR concentration was determined by a validated LC-MS / MS method.

[0154] Briefly, the triple quadrupole LC-MS / MS system consisted of a 1200 series HPLC system (Agilent Technologies, USA) and a mass spectrometer (6420 triple quadrupole LC / MS, Agilent Technologies, USA). Chromatographic separation was achieved on a Waters Xbridge-C18 (2.1 mm × 50 mm, 3.5 μm) column at 40 °C, with isocratic mobile phase A being an aqueous solution of 10 mM ammonium acetate and 0.1% formic acid, and mobile phase B being acetonitrile.

[0155] The gradient was 0 min, 10% B; 0.8 min, 10% B; 2 min, 20% B; 3.0 min, 90% B; 3.5 min, 90% B; then returned to 10% B within 0.5 min and equilibrated for 0.8 min for the next injection. The injection volume was 10 μL and the flow rate was 0.2 mL / min. Both VCR and vinblastine (as the internal standard) were ionized by the ESI source in positive ion mode. The mass spectrometry parameters were as follows: capillary, 5000 V; gas temperature, 320 °C; gas flow, 8 L / min; and nebulizer, 40 psi. The transition of m / z 825→765 was quantified using multiple reaction monitoring (MRM), where the collision energy (CE) of VCR was 40 eV and the fragmentation voltage was 280 V; the collision energy (CE) of vinblastine for m / z 811→355 was 40 eV and the fragmentation voltage was 280 V. System control and data analysis were performed using MassHunter workstation software for qualitative analysis (version B.06.00) and quantitative analysis (version B.05.02).

[0156] Although VCR has good anti-cancer activity, its effectiveness in brain tumors is limited due to its inability to penetrate the BBB / BBTB and dose-limiting neurotoxicity. Therefore, STICK-NPs were used to deliver VCR, and their anti-cancer effects were evaluated in a very aggressive and invasive orthotopic DIPG brain tumor model. An orthotopic model was established by injecting pediatric DIPG cells into the pons of SCID mouse brains. After confirmation of the establishment of DIPG brain tumors in mice using Gd-enhanced T1-weighted MRI ( Figure 6 a), the mice were randomly divided into 9 groups: PBS, 1.5 mg / kg free VCR, NM@VCR, MA-NP@VCR, CBA-NP@VCR, STICK-NP@VCR, and vincristine sulfate liposomes (Marqibo, liposomal VCR), and two high-dose groups, free VCR2 and STICK-NP@VCR2 (VCR 2 mg / mL) (n = 6). Since this is a very aggressive DIPG model, free VCR (1.5 and 2 mg / kg), NM@VCR, MA-NP@VCR, CBA-NP@VCR, and vincristine sulfate liposomes (Marqibo) had little inhibitory effect on tumor growth and failed to extend the survival time of the animals compared to the PBS control ( Figure 6 a-6d). Very encouragingly, STICK-NP@VCR showed promising effects in hindering tumor growth ( Figure 6 a-6c; Figure 13 ), with the survival time almost doubling (21.3 days) compared to vincristine sulfate liposomes, CBA-NP@VCR, and MA-NP@VCR (survival times of 12.5 days, 12 days, and 12 days, respectively) (as Figure 6 d). Even at a higher dose (2 mg / kg), VCR did not benefit the survival time of DIPG mice (as Figure 6 a-6c). In contrast, STICK-NP@VCR at an equivalent dose level further extended the overall survival time, and 2 out of 6 mice in this group survived for more than 50 days. To achieve the best effect, the remaining animals were treated continuously with 2 mg / g STICK-NP@VCR every 6 days. The orthotopic DIPG tumors in these mice were completely eradicated. During the treatment, the body weight did not change significantly until the development of neurological syndromes due to increased tumor burden and invasion (as Figure 6 e; as Figure 14 ). In addition, a similar efficacy study was conducted in an orthotopic GBM model with a higher degree of vascularization in nude mice (as Figure 15 ). STICK-NP@VCR was consistently superior to other formulations at a single dose of 2 mg / kg VCR. Based on MRI and histopathology (as Figure 15a, 15d), STICK@VCR significantly inhibited tumor progression and prolonged the median survival time (34 days) compared to other formulations (all less than 17 days). Major organs were collected on day 12 after treatment, and no significant pathological changes were found in all groups ( Figure 15 f). STICK-NPs can effectively deliver high doses of chemotherapeutic drugs to the tumor site and eradicate brain tumors with limited toxicity. The disappointing anticancer results of CBA or MA single-targeted nanoparticles again indicate the need to consider the complexity and dynamic environment during brain tumor delivery.

[0157] Example 4. Treating Diseases

[0158] Cell culture: Mouse bEnd.3 cells and human U87-MG cells were obtained from ATCC and maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C in a 5% CO2 environment. U87-MG cells were transfected with GFP for imaging studies.

[0159] Membrane filter Culture system: To mimic the BBB / BBTB, a membrane filter was used to culture bEnd.3 cells in the upper chamber and U87-MG with or without culture in the lower chamber. The pore size of the membrane filter (Transwell) was 0.4 μm, and 5×104 bEnd.3 cells were seeded in each well. The integrity of the bEnd.3 monolayer in vitro was evaluated by transendothelial electrical resistance. After 7 days, the transendothelial electrical resistance value reached 200 Ω·cm 2 or more, and it was considered that tight junctions were formed. Then, U-87-MG cells were cultured in the lower chamber overnight. STICK-NP@Cy (0.2 mg / mL Cy7.5) and other shown controls were placed in the upper chamber for 2 hours to allow spontaneous transcytosis. Samples in the lower chamber were collected at different time points to detect Cy fluorescence and particle size using DLS (using PBS instead of FBS). The membrane filter (Transwell) was removed, and the pH of the lower chamber medium was adjusted to pH 6.5 with 10 mM HCl or maintained at pH 7.4. The medium containing nanoparticles was further left with U87-MG cells in the lower chamber for another hour to allow intracellular uptake. The intracellular uptake of U87-MG cells in the lower chamber was monitored using a fluorescence microscope (BZ-X700, Keyence, Japan). The imaging was quantified and analyzed by Image J.

[0160] In vitro and in vivo permeability studies: The second obstacle encountered by STICK-NPs is the BBB / BBTB, the tight junctions formed by brain microvascular endothelial cells. The excessive proportion of MA (glucose derivative) on STICK-NPs is the first exposed targeting group for GLUT1-mediated transcytosis by endothelial cells, while CBA is covered in STICK( Figure 1 a). Mouse brain endothelial cells (bEnd.3) were cultured in the apical chamber of the membrane filter system, and the formation of tight junctions was confirmed by transendothelial electrical resistance (TEER) > 200 Ω.cm 2 (as Figure 3 a). At different time points after loading the nanoparticles into the apical chamber, the total fluorescence intensity of the culture medium in bEnd.3 cells (during transcytosis) (as Figure 3 b, Figure 10 ) and the basal chamber (after transcytosis) (as Figure 3 c) was evaluated. Figure 3 b shows that STICK-NP@Cy and MA-NP@Cy (also targeting GLUT1 through MA) have the highest intracellular signal in all groups. Consistent with this finding, the STICK-NP@Cy and MA-NP@Cy groups have the highest lateral vector of tight junctions entering the basal chamber (as Figure 3 c). When GLUT1 was blocked by the GLUT1 inhibitor (WZB-117) (as Figure 9 a-9b), the lateral movement of STICK-NP@Cy decreased. The most interesting finding is that when comparing the sizes of STICK-NP@Cy in the apical and basal chambers, the size of STICK-NP@Cy remained similar before (~164 nm) and after (~146 nm) transcytosis by bEnd.3 cells (as Figure 3 d). When evaluating the subcellular distribution of STICK-NP@DiD in bEnd.3 cells, it was found that STICK-NP@DiD did not co-localize with lysosomes, with a low Pearson's coefficient index of 0.057. It is speculated that if the lysosome-dependent pathway occurred, the low lysosomal pH value (5.5) should have disrupted the crosslinking and initiated the release of secondary smaller micelles. These combined evidences support the view that STICK-NPs may cross the BBB through the transcytosis pathway, and further detailed mechanism studies are underway.

[0161] U87-MG three-dimensional spheres were cultured according to the reported method. Briefly, U87-MG-GFP cells were seeded at 1×10 4Cells were seeded at a density of

[0162] An orthotopic brain tumor model was established by injecting 2.5 × 104 DIPG (PDX) cells into the left side of the brainstem of female SCID mice. Mice were injected with STICK-NP@DiD and NM@DiD (DiD at 2.5 mg / kg). After 24 h, the mice were sacrificed and FITC-dextran (70K) was injected 2 min before sacrifice to label blood vessels.

[0163] Cell uptake analysis: Finally, after crossing the BBB, STICK-NPs enter the acidic tumor microenvironment (barrier 3). In response to the lower extracellular pH, STICK is degraded, leading to the release of secondary micelles ( Figure 3 d, 3g). CBA was initially covered as part of STICK and is now exposed after crosslinking cleavage as a secondary tumor targeting group for brain tumors ( Figure 1 a and Figure 3 g). Next, the brain tumor cell targeting and cell uptake capabilities of secondary STICK-NPs were investigated using fluorescence imaging. Human U87-MG GBM cells were treated with STICK-NP@Cy and other control formulations at pH 7.4 and pH 6.5 for 4 h ( Figure 3 h - 3i). The results showed that the total cell uptake of all groups was relatively low at pH 7.4, including STICK-NPs with covered CBA. In contrast, pretreatment at pH 6.5 to expose CBA significantly enhanced the uptake of STICK-NP@Cy by brain tumor cells. Conversely, even with pretreatment at pH 6.5, the free Cy7.5, MA-NP@Cy, CBA-NP@Cy, and NM@Cy groups did not show significant enhancement. To further explore the potential role of sialic acid expression in nanoparticle uptake, cells were treated with 3-azido thymidine (AZT) to increase surface sialic acid expression. This treatment further promoted the uptake of STICK-NPs (pH 6.5) by tumor cells. Figure 3 h - 3j). Additionally, the cell uptake of CBA-mediated STICK-NPs (pH 6.5) could be completely blocked by an excess of free CBA ( Figure 3h-3j). These results demonstrate that STICK-NPs can be effectively internalized by transformed brain tumor cells, which may be due to the newly discovered CBA enhancing sialic acid-mediated transcytosis. It is worth considering that at pH 6.5, the affinity of CBA for sialic acid is much higher than that for glucose (in terms of MA), so it is preferably bound to sialic acid on tumor cells.

[0164] To investigate the cellular uptake of STICK-NP@Cy, bEnd.3 cells or U87-MG cells were seeded on 8-well chamber slides (10,000 cells / well) and treated with STICK-NP@Cy and other controls (0.1 mg / mL Cy7.5) for 1 hour and washed three times with PBS. Then the cells were fixed and stained with DAPI. Cellular imaging was obtained using a Keyence fluorescence microscope. For quantitative studies, bEnd.3 cells or U87-MG cells (10,000 cells / well) were seeded in 96-well plates overnight and then treated with STICK-NP@Cy and other controls (0.1 mg / mL Cy7.5). Cells were harvested at 0 h, 1 h, 2 h, 3 h, and 4 h and washed with PBS. All cells were lysed with 100 μL DMSO, and the fluorescence intensity was measured by a fluorescence spectrophotometer (RF-6000, Shimadzu, Japan). To inhibit GLUT1 activity, bEnd.3 cells were pretreated with 40 μM WZB-117 for 24 hours and then incubated with STICK-NP@Cy. For tumor uptake studies, U87-MG cells were pretreated with 40 μM AZT for 24 hours to alter the surface sialic acid expression. To block the interaction, U87-MG cells were pre-incubated with excess free CBA (80 μM) for 24 hours to compete for the binding site with the CBA targeting group in the secondary smaller micelles of STICK-NP@Cy (pH 6.5).

[0165] To simulate the combination of Barrier 2 (BBB / BBTB) and Barrier 3 (brain tumor uptake) upon delivery to brain tumors, bEnd.3 cells were cultured in the upper chamber of a membrane filter (Transwell), and U87-MG brain tumor cells were cultured in the lower chamber (as Figure 3 k). STICK-NP@Cy and other control NPs were loaded into the upper chamber for 1 hour, and the pH of the lower chamber medium was adjusted to 7.4 or 6.5 and maintained for another 1 hour to allow U87-MG tumor cells to uptake. As expected, as Figure 3As shown in l and m, compared with the STICK-NP@Cy (pH7.4), MA-NP@Cy, CBA-NP@Cy, and NM@Cy (pH7.4 and 6.5) groups or the free dye in the lower chamber, the STICK-NP@Cy (pH6.5) group achieved the highest uptake in U87-MG cells. GLUT1 inhibition also hindered the final U87-MG cell uptake, which may be due to reduced transcytosis ( Figure 3 b-3c). These results together provide step-by-step verification for the mechanism by which STICK-NP significantly enhances drug delivery, including BBB / BBTB transcytosis, transformation, and tumor cell uptake. Importantly, single-targeted nanoparticles with CBA or MA may slightly improve delivery to brain tumors, but the efficiency is still suboptimal compared to STICK-NP.

[0166] After transcytosis and transformation, STICK-NPs release a large number of secondary micelles with a diameter of approximately 20 nm, which are more suitable for deep tissue penetration within the tumor ( Figure 1 b, 1d). The three-dimensional multicellular spheroid system most closely resembles in vivo conditions and forms a compact extracellular matrix environment, allowing for testing of drug penetration in vitro. To evaluate the size-related tissue penetration effect, U87-MG neurospheres (∼400 μm) were incubated with STICK-NP@DiD and other control formulations at pH 7.4 or 6.5. After 24 hours, confocal fluorescence imaging of U87-MG spheroids showed that due to its relatively large size (∼142 nm) ( Figure 1 b), the untransformed STICK-NP@DiD (pH 7.4) group had poor penetration and a lower penetration depth (30.1 μm ± 5.9 μm) ( Figure 4 a; Figure 11 ). In pH-dependent transformation, STICK-NP@DiD (pH 6.5) had significantly superior penetration ability compared to STICK-NP@DiD (pH 7.4), and reached a similar depth compared to other small-sized (∼20 nm) nanopreparations (such as Figure 4 a; such as Figure 1 b; such as Figure 11 ). A similar pH-dependent transformation / penetration effect was further confirmed in DIPG human tissue xenograft (PDX) neurospheres (diameter ∼300 μm) ( Figure 4 b). The pH-responsive property actually endows STICK-NP with tumor selectivity. Therefore, an in situ DIPG model was used to evaluate the tissue penetration extent of STICK-NPs in normal brain and acidic tumor sites. Figure 4c-4d showed that at 24 hours, STICK-NP@DiD was able to penetrate DIPG tumor tissue approximately 30 μm away from blood vessels. In contrast, in normal cerebral parenchyma (reported pH value of canine cerebral parenchyma was 7.13), STICK-NP@DiD penetrated only approximately 5 μm outside blood vessels. Meanwhile, the NM@DiD control had the least normal brain penetration ability ( Figure 4 c). Together with in vitro studies, the conclusion was drawn that STICK-NP could selectively respond to the acidic environment to release secondary nanoparticles with newly discovered CBA targeting groups, thus better penetrating tumor tissue and tumor cell uptake. Due to its pH selectivity, STICK-NP had limited penetration ability into normal tissues and less concern about neurotoxicity.

[0167] Anticancer efficacy study in orthotopic brain tumor models: As described above, orthotopic brain tumor models were established by injecting 2.5×104 DIPG (PDX) cells into the left side of the brainstem of female SCID mice or 5×104 GBM (U87-MG) cells into the left side of the brain of female nude mice. After confirming the establishment of brain tumors, the mice were randomly assigned to different groups. The tumor size was monitored using advanced T1-weighted imaging (TR / TE = 300 ms / 15 ms). For imaging studies, the mice were injected with 250 mg / kg Gd-DTPA. The tumor size in the DIPG model was calculated from the aggregation of the tumor area in different MRI slices (1 mm thick). The tumor size in the GBM model was calculated using the following formula:

[0168]

[0169] where W was the width of the tumor and L was the length of the tumor (W < L). On the 12th day after MRI imaging, one mouse from each group was sacrificed, and the organs and brains with tumors were collected for histopathological evaluation. The appearance, behavior, and body weight of the animals were continuously monitored. Once the weight loss > 20%, the animal was considered to reach the humane endpoint.

[0170] The targeted delivery of STICK-NPs was further investigated in the orthotopic DIPG PDX model. Gd-enhanced T1-weighted MRI was first used to localize DIPG. After the Gd signal cleared, the mice were reinjected with DiD+Gd, NM@DiD+Gd, and STICK-NPs@Gd@DiD, and re-imaged 16 hours after injection ( Figure 5 f). As Figure 5As shown in f, STICK-NPs@Gd@DiD selectively and effectively accumulate in the tumor site, as shown by both imaging modalities. Imaging studies strongly demonstrate that STICK-NP@Cy@Gd can specifically deliver payloads to the tumor site, thus allowing for accurate image-guided drug delivery and potential use in delineating tumor margins during surgery. In contrast, the single-targeting formulations MA-NP and CBA-NPs, which showed targeting effects in vitro previously, failed to deliver sufficient payloads to orthotopic brain tumors in vivo.

[0171] Example 5. Imaging

[0172] ARS-based fluorescence analysis: ARS is a catechol dye, and its absorption and fluorescence intensity change significantly after binding to boric acid. ARS-based fluorescence analysis was used to confirm the formation of borate ester bonds in solution. Briefly, ARS (0.1 mg / mL) was mixed with CBA4-PEG-CA8 (2.5 μM) and different concentrations of MA4-PEG-CA8 (0 - 40 μM). The change in the fluorescence intensity of ARS (Em: 585 nm, Ex: 468 nm) was measured using a fluorescence spectrophotometer (Shimadzu, RF-6000).

[0173] Establishment of orthotopic brain models and optical and magnetic resonance imaging studies: Next, the orthotopic PDX GBM model was used to evaluate the biodistribution of STICK-NPs@Cy@Gd using dual-modal imaging: NIRF imaging (Cy7.5) and MRI (Gd-DTPA)( Figure 5 a). At 10 minutes post-injection, the whole-brain MRI T1-weighted signal increased in all groups( Figure 5 a). At 24 and 48 hours post-injection, compared with the free Cy7.5+Gd, NM@Cy+Gd, CBA-NP@Cy+Gd, and MA-NP@Cy+Gd groups, the STICK-NP@Cy@Gd group had significantly higher T1-weighted MRI signal intensities( Figure 5 a-5b) and Cy7.5 fluorescence intensities( Figure 5 a, 5c, 5d) at the tumor site. It should be noted that, unlike STICK-NPs, the hydrophilic Gd-DTPA cannot be loaded into NM, CBA-NPs, and MA-NPs, so in these groups, it was injected as free Gd-DTPA together with the nanoparticles loaded with Cy7.5 as a control group. The NIRF or T1-weighted MRI signal of STICK-NP@Cy@Gd remained the longest in the tumor and only returned to baseline 72 hours after injection( Figure 12a). Although only 1 / 3 of the clinical dose of Gd-DTPA was used, this specific PDX model seemed to exhibit poor permeability, as demonstrated by the minimal T1 signal of Gd-DTPA at the tumor site at 10 minutes ( Figure 5 a). Nevertheless, STICK-NPs could still effectively target, penetrate, and retain in the PDX GBM model.

[0174] To further dissect the targeting delivery efficiency and selectivity for entering brain tumors, another group of mice was sacrificed 24 hours after nanoparticle administration, and the major organs / brains with brain tumors were harvested for ex vivo NIRF imaging. The biodistribution was evaluated based on the Cy7.5 signal in the brain and other major organs. As Figure 5 shown in a, 5d, 12b, and 12c, compared with other major organs except the kidney, STICK-NPs could specifically deliver a higher concentration of Cy7.5 to the orthotopic PDX GBM tumor, which might be the clearance pathway of the Cy7.5 dye. Compared with free Cy7.5+Gd and NM@Cy+Gd, the accumulation of the Cy7.5 signal at the brain tumor site in the STICK-NPs treatment group was significantly higher. NIRF imaging of cryosections of orthotopic brain tumors in the STICK-NPs group showed a strong correlation between tumor cells (green) and Cy7.5 (red) ( Figure 5 e; Figure 12 d), and the calculated Pearson's coefficient index was as high as 0.637. Meanwhile, the uptake in the normal brain was minimal, indicating excellent tumor selectivity of STICK-NPs ( Figure 5 c, 5e). Semi-quantitative imaging analysis showed that in MRI and NIRF imaging, the signals of orthotopic glioblastoma PDX tumors were 1.5 times and 4 times those of the adjacent normal brain tissue, respectively ( Figure 5 b, 5d).

[0175] Human-derived tissue xenograft (PDX) glioblastoma was kindly provided by Dr. C. David James of the University of California, San Francisco. The cells were first transfected with GFP. To establish orthotopic brain tumors, with the help of a mouse stereotaxic apparatus (Stoelting), 5 μl of PDX cells (1x10 7 / mL) or U87 (1x10 7(in mL) was injected into the right striatal region of the mice. The cells were injected within 5 minutes, and the mice were allowed to rest for another 5 minutes under general anesthesia. The animals received pain management for 3 days postoperatively. Two weeks later, the animals were intravenously injected with STICK-NP@Cy@Gd and the other control groups shown (Cy7.5: 10 mg / kg; Gd: 25 mg / kg). In vivo near-infrared red fluorescence imaging was obtained at different time points as shown using a Kodak imaging station (4000MM). The same mice also received T1-weighted MR imaging of the brain at 0 minutes, 10 minutes, 24 hours, 48 hours, and 72 hours. A Bruker BioSpec 7T MRI scanner was used to record the imaging through coronal cross-sectional views. The following parameters were used for all T1-weighted MR images recorded: TR = 400 ms; TE = 15 ms; matrix = 256x256; FOV = 20x20 mm2. Twenty-four hours after imaging, the mice were sacrificed, and all organs, including the brain containing the tumor, were harvested for ex vivo imaging. The whole brain with the tumor was fixed in a compound of optimal cutting temperature. 10-μm cryosections were used for fluorescence microscopy imaging (Keyence), and the cell nuclei were stained with DAPI.

[0176] In summary, the STICK technology provides a simple and intelligent solution to address multiple obstacles in drug delivery to brain tumors. STICK is designed based on a unique pair of two targeting groups that can also form stimulus-responsive bonds, such as glucose derivatives and the borate family that can form pH-responsive borate ester crosslinks. In the current STICK approach, the targeting groups (CBA or MA) serve far more than just a targeting purpose. They are integrated into the nanoparticle structure and significantly contribute to the ideal properties (such as stability, stimulus responsiveness, transformability, and multifunctional drug-loading capacity) of these nanoparticles and the overall delivery performance. This unique STICK design is significantly different from the previously reported dual-targeting systems. The STICK strategy was introduced into a well-characterized micellar formulation, and it was shown that STICK-NPs could be preserved in the bloodstream and STICK sequentially entered the BBB / BBTB and brain tumor cells respectively. STICK-NPs were demonstrated to overcome the unstable environment in the blood through the intermicellar crosslinking formed by MA (exposed) and CBA (covered), and showed a significantly extended circulation time, thus allowing a wider brain tumor targeting window ( Figure 1 ). During circulation, the excessive MA on the nanoparticle surface can promote GLUT1-mediated endocytosis through the BBB / BBTB, thus "actively" targeting brain tumors ( Figure 3 ). Subsequently, upon encountering the inherent acidic pH value at the tumor site, STICK breaks, triggering the transformation into secondary smaller nanoparticles for in-depth penetration of the tumor tissue ( Figure 4), and exposes the secondary targeting group, i.e., CBA targeting sialic acid overexpressed in tumor cells, to enhance cellular uptake( Figure 5 ). The pH-dependent selectivity further endows them with biosafety characteristics. In orthotopic glioblastoma and DIPG mouse models, STICK-NPs effectively deliver hydrophobic and hydrophilic contrast agents to the tumor site for bimodal imaging. Most excitingly, compared with single-targeting formulations, even in the most aggressive and VCR-resistant DIPG model, STICK-NP@VCR also exhibits excellent brain tumor suppression effects and significantly extended survival times( Figure 6 ). These promising results highlight the unique features of STICK in overcoming different complex barriers, as well as the importance of considering all obstacles during nanoparticle design for successful delivery to brain tumors. Given the multifunctional drug-loading capacity, STICK-NP can offer a direct second hope for delivering state-of-the-art epigenetic modulators, such as HDAC and EZH2 inhibitors, whose efficacy is greatly hindered by the BBB / BBTB, leading to clinical trial failures. The STICK strategy provides notable opportunities for applying this approach to many other nanoparticle designs to combat dynamic and entangled biological barriers, and also has an impact on advancing drug development / delivery against aggressive brain tumors.

[0177] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clear understanding, those skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. Additionally, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were incorporated by reference individually. In the event of a conflict between the present application and the references provided herein, the present application shall govern.

Claims

1. A compound as described by formula (Ib), Wherein: Each R 1 is maltobionic acid; Each R 2 is cholic acid; each X is lysine; and the PEG has a molecular weight of 5 kDa.

2. A compound as described by formula (Ib), Wherein: Each R 1 is 4-carboxyphenylboronic acid; Each R 2 is cholic acid; each X is lysine; and the PEG has a molecular weight of 5 kDa.

3. A nanoparticle, characterized in that it comprises a plurality of first and second conjugates, wherein: each first conjugate is the compound according to claim 1; each second conjugate is the compound according to claim 2; and the plurality of first and second conjugates self-assemble by forming crosslinking bonds to form a nanoparticle, such that the interior of the nanoparticle comprises a hydrophilic interior, the hydrophilic interior comprising a plurality of micelles having a hydrophobic core; and the ratio of the first conjugate to the second conjugate is 10:1, 9:1, or 5:

1.

4. A nanoparticle comprising a hydrophilic exterior and interior, characterized in that the interior of the nanoparticle comprises a hydrophilic interior, the hydrophilic interior comprising a plurality of micelles, wherein each micelle comprises a hydrophobic core, a hydrophilic exterior, and a plurality of first and second conjugates, wherein: each first conjugate is the compound according to claim 1; each second conjugate is the compound according to claim 2; and the plurality of first and second conjugates self-assemble by forming crosslinking bonds to form a micelle having a hydrophobic core, wherein the crosslinking bonds are in the hydrophilic exterior of the micelle; and the ratio of the first conjugate to the second conjugate is 10:1, 9:1, or 5:

1.

5. The nanoparticle according to any one of claims 3 to 4, characterized in that the nanoparticle further comprises a hydrophilic drug or a contrast agent.

6. The nanoparticle according to claim 5, characterized in that the hydrophilic drug or contrast agent is gadopentetic acid (Gd-DTPA), indocyanine green (ICG), cisplatin, gemcitabine, doxorubicin hydrochloride (DOX×HCl) or cyclophosphamide.

7. The nanoparticle according to any one of claims 3 to 4, characterized in that the nanoparticle further comprises a hydrophobic drug or a contrast agent.

8. The nanoparticle according to claim 7, characterized in that The hydrophobic drug or contrast agent is Cy7.5, DiD, doxorubicin (DOX), vincristine (VCR), everolimus, carmustine, lomustine, temozolomide, lenvatinib mesylate, sorafenib tosylate, regorafenib, irinotecan, paclitaxel (PTX), docetaxel, BET inhibitor, OTX015, ABBV-075, HDAC inhibitor, valproic acid, vorinostat, panobinostat, entinostat, ricolinostat, AR-42, JMJD3 inhibitor, GSKJ4, EZH2 inhibitor, tazemetostat, GSK2816126, MC3629, EGFR inhibitor, gefitinib, erlotinib, lapatinib, osimertinib, IDH inhibitor, enasidenib, ivosidenib, Notch inhibitor, RO4929097, CDK4 / 6 inhibitor, palbociclib, ribociclib, abemaciclib, PI3K / Akt / mTOR inhibitor, rapamycin, buparlisib, curcumin or etoposide.

9. The nanoparticle according to claim 7, wherein, the hydrophobic drug or contrast agent is BET-d246, I-BET151, or I-BET 762.

10. The nanoparticle according to any one of claims 3 to 4, wherein, the ratio of the first conjugate to the second conjugate is 9:

1.

11. Use of a nanoparticle according to any one of claims 3 to 10 for the preparation of a medicament for delivery, wherein, the nanoparticle further comprises a hydrophilic and / or hydrophobic drug and a plurality of crosslinking bonds; and wherein the crosslinking bonds are cleavable in situ such that the drug is released from the nanoparticle, thereby delivering the drug to a subject in need thereof.

12. The use according to claim 11, wherein, the hydrophilic and / or hydrophobic drug is doxorubicin hydrochloride (DOX×HCl), doxorubicin (DOX), vincristine (VCR) or paclitaxel (PTX).

13. The use according to claim 11 or 12, wherein, the medicament is a medicament for treating a disease selected from the group consisting of glioblastoma, diffuse pontine glioma, brain metastases, lung cancer, breast cancer, colon cancer, kidney cancer, or melanoma.

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