PH-responsive compositions and uses thereof

Abstract

The invention relates to a pH-responsive composition and a use thereof. Described herein are pH-responsive compounds, micelles, and compositions useful for the detection of primary and metastatic tumor tissue. The compounds described herein are imaging agents useful for the detection of primary and metastatic tumor tissues, including lymph nodes. Real-time fluorescence imaging during surgery helps a surgeon to detect metastatic lymph nodes or depict tumor tissue relative to normal tissue, with the goal of achieving negative marginal incision and complete tumor resection.

Description

[0001] This application is a divisional application of Chinese patent application filed on May 28, 2020, with application number “202080053111.2” and invention title “pH-responsive composition and its use”. The original application was the Chinese national phase application of international application PCT / US2020 / 034783.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 853,593, filed May 28, 2019, which is incorporated herein by reference in its entirety.

[0004] Statement on Federally Funded Research

[0005] This invention was made with the support of the U.S. government under National Institutes of Health (NIH) licenses R01 EB 013149 and CA 192221. Technical Field

[0006] This invention relates to pH-responsive compositions and their uses. Background Technology

[0007] In 2019, an estimated 1.7 million new cancer cases were diagnosed, and approximately 610,000 Americans were expected to die from cancer. Effective imaging agents are needed to detect primary and metastatic tumor tissue.

[0008] Treatment guidelines for solid cancers at all stages primarily involve surgical removal of the primary tumor and at-risk or involved lymph nodes. Despite biological and anatomical differences among these tumor types, postoperative margin status is one of the most important prognostic factors for local tumor control and therefore a chance of recurrence or metastasis. Surgical resection of solid tumors is a balance between oncologic efficacy and minimization of normal tissue removal, and thus involves functional pathogenesis. This also applies to lymph node dissection for diagnostic and therapeutic purposes, which is often performed concurrently with the removal of the primary cancer. The presence or absence of lymph node metastasis is a crucial survival determinant for many solid cancers.

[0009] Optical imaging strategies, based on cell imaging, natural autofluorescence, and Raman scattering, have been rapidly adapted for intraoperative tissue imaging. The potential of optical imaging includes real-time feedback and availability of the imaging system, providing a wide surgical field of view. One strategy to overcome the complexities encountered during surgery due to the diversity of oncogenotypes and histological phenotypes is to target the pervasive metabolic vulnerability in cancer. Aerobic glycolysis (known as the Warburg effect) occurs in all solid cancers, where cancer cells preferentially take up glucose and convert it to lactate.

[0010] Therefore, there is still a need to develop compositions and methods for identifying the presence of cancer (especially cancer metastasis) in the lymphatic system. Summary of the Invention

[0011] The block copolymers proposed in this paper utilize the prevalent pH difference between cancerous and normal tissues and provide a highly sensitive and specific fluorescence response after being taken up by cells, thereby allowing the detection of tumor tissue, tumor margins, and metastatic tumors (including lymph nodes).

[0012] The compounds described in this article are imaging agents that can be used to detect primary and metastatic tumor tissue, including lymph nodes. Real-time fluorescence imaging during surgery helps surgeons detect metastatic lymph nodes or delineate tumor tissue relative to normal tissue, with the goal of achieving negative surgical margins and complete tumor resection. Clinical benefits from improved surgical outcomes include, for example, reduced tumor recurrence and reoperation rates, avoidance of unnecessary surgeries, and informed patient treatment plans.

[0013] In some embodiments, block copolymers of formula (I), or pharmaceutically acceptable salts, solvates, or hydrates thereof, are provided herein:

[0014]

[0015] Where: n is 113; x is 60 to 150; y is 0.5 to 1.5, and R' is a halogen, -OH or -C(O)OH.

[0016] In some embodiments, micelles are provided herein that comprise one or more of a formula (I) block copolymer or a pharmaceutically acceptable salt, solvate, hydrate or isotopic variant thereof.

[0017] In some embodiments, pH-responsive compositions are provided herein comprising micelles of a block copolymer of formula (I), wherein said micelles have a pH transition point and an emission spectrum. In some embodiments, the pH transition point is 4 to 8. In some embodiments, the pH transition point is 6 to 7.5. In some embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the pH transition range (ΔpH) is... 10至90% The pH value is less than 1 pH unit. In some embodiments, the emission spectrum is 700 to 850 nm. In some embodiments, the pH transition range (ΔpH) is... 10至90% The pH value is less than 0.25 pH units. In some embodiments, the emission spectrum is 700 to 850 nm. In some embodiments, the pH transition range (ΔpH) is... 10至90% (less than 0.15 pH units)

[0018] In some embodiments, methods for imaging the pH of an intracellular or extracellular environment are provided herein, comprising: (a) contacting a pH-responsive composition of the present disclosure with an environment; and (b) detecting one or more light signals from the environment, wherein detection of the light signals indicates that micelles have reached their pH transition point and dissociated. In some embodiments, the light signals are fluorescent signals. In some embodiments, imaging of the intracellular environment involves contacting cells with a pH-responsive composition under conditions suitable for inducing uptake of the pH-responsive composition. In some embodiments, the intracellular environment is part of a cell. In some embodiments, the extracellular environment is the extracellular environment of tumor or vascular cells. In some embodiments, the extracellular environment is intravascular or extravascular. In some embodiments, the tumor is cancer, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer. In some embodiments, the tumor is metastatic tumor cells. In some implementations, metastatic tumor cells are located in lymph nodes.

[0019] Other objects, features, and advantages of the compounds, methods, and compositions described herein will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples indicate particular embodiments, they are given by way of example only, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art based on this detailed description.

[0020] By incorporating via reference

[0021] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0022] Figures 1A to 1D The binary fluorescence response of the ultra-pH sensitive (UPS) polymer micelle probe is shown. Figure 1A UPS micelles are self-assembled nanoparticles that break down into monomers in response to a threshold proton concentration. Figure 1B The structure of the amphiphilic block copolymer enables a synergistic pH response at a specific pKa. Figure 1C Dynamic light scattering revealed different size populations of monomers (pH below pKa) used for USP6.1. Figure 1D The nonlinear amplification of fluorescence intensity reveals an ultra-pH-sensitive response to ambient pH signals. Near-infrared visualization of UPS5.3-ICG (top), UPS6.1-ICG (middle), and UPS6.9-ICG (bottom) as a function of pH is shown in the inset tube.

[0023] Figures 2A to 2C show the in vitro characterization of UPS-ICG nanoparticles. (Figure 2A) UPS-ICG nanoparticles at a wavelength of 788 nm. 最大 It absorbs near-infrared light. (Figure 2B) Raw mean fluorescence intensity of UPS-ICG nanoparticles measured by LI-COR Pearl 800 nm channel. (Figure 2C) Number mean diameter of UPS-ICG nanoparticles measured by dynamic light scattering.

[0024] Figures 3A to 3D show whole-body near-infrared fluorescence imaging of dissected, tumor-naïve BALB / cj mice, enabling image-guided resection of liver nuclei (LNs) in real time. (Figure 3A) UPS5.3-ICG and (Figure 3B) UPS6.1-ICG depict all superficial LNs, allowing for image-guided resection. (Figure 3C) UPS6.9-ICG fluorescence primarily sequesters the liver. Image-guided resection of LNs is not permitted in these cases. (Figure 3D) Median fluorescence intensity of LNs is normalized relative to median fluorescence intensity of skeletal muscle (Mu). The median CR of the dissected LN groups shows dependence on the pKa of polymeric micelles. UPS5.3 shows the highest intensity among all LN dissection groups.

[0025] Figures 4A through 4C illustrate the pharmacokinetics and organ distribution of UPS nanoparticles in Balb / cj mice. (Figure 4A) Pharmacokinetics of UPS-ICG fluorescence in collected plasma. Plasma was acidified to show the “ON” state of the nanoparticles. Plasma fluorescence was normalized relative to fluorescence at time 0 hours, controlling for differences between UPS compositions. (Figure 4B) Normalized acidified plasma fluorescence relative to collected plasma, showing the “ON / OFF Ratio”. (Figure 4C) Ex vivo imaging of organs after 24 hours of UPS nanoparticle circulation.

[0026] Figures 5A to 5C illustrate the colocalization of UPS nanoparticles with macrophage subsets, showing the uptake of micelles by lymph node-resident macrophages. (Figure 5A) UPS5.3-ICG colocalizes with CD169 (left), F4 / 80 (middle), and CD11b (right), but this colocalization is limited to within the lymph node. White arrows indicate colocalization between positive cells and ICG fluorescence. Light gray arrows indicate staining of F4 / 80 cells in the absence of ICG fluorescence. (Figure 5B) The pattern of colocalization of UPS6.1-ICG with macrophages mirrors that of UPS5.3-ICG. (Figure 5C) The fluorescence intensity of UPS6.9-ICG is significantly lower than that of UPS5.3-ICG and UPS6.1-ICG. All figures demonstrate the phagocytosis of nanoparticles by macrophages in the lymph node (rather than macrophages in surrounding tissues). Scale bar is 200 µm.

[0027] Figures 6A through 6F show the detection of metastatic lymph nodes as verified by histological examination. (Figure 6A) Representative 4T1.2-load BALB / cj mice administered UPS5.3-ICG show primary tumors (PT) and LNs depicted as benign (Be), micrometastatic (Mi), and macrometastatic (Ma) by NIRF on whole-body imaging, enabling image-guided resection of inguinal (In), axillary (Ax), and cervical (Cr) LNs. (Figure 6B) NIRF imaging of mice administered UPS6.1-ICG shows the depiction of primary tumors and LNs, where benign LNs appear almost as bright as metastatic LNs. (Figure 6C) UPS6.9-ICG accumulates in the liver (Li) with a much higher intensity. Some large metastatic LNs were depicted, but many micrometastatic LNs were undetectable. (Figure 6D) The UPS5.3 signal and median CR of the categorical tissue showed significance between metastatic and benign LNs. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test (*P < 0.033, **P < 0.0021, ***P < 0.0002, ****P < 0.0001). (Figure 6E) The UPS6.1 signal and median CR of the categorical tissue showed significance between large metastatic and benign LNs, but the variance in the large metastatic distribution was high. (Figure 6F) The UPS6.9 signal and median CR of the categorical tissue showed significance between large metastatic and benign LNs. The strength of the signal variable was much lower compared to UPS5.3 and UPS6.1.

[0028] Figures 7A and 7B This study demonstrates the real-time resection of metastatic lymph nodes guided by NIR fluorescence. (Fig. 7A) BALB / cj mice bearing 4T1.2 were intravenously injected with UPS5.3-ICG, euthanized, dissected, and imaged at 4 fps using a near-infrared camera. All superficial LNs and primary tumors were depicted. (Fig. 7B) LNs are visible in the anatomical region. Large metastatic LNs showed increased fluorescence intensity, significant spatial accumulation of fluorescence, and were larger than other LNs. This LN was resected using NIR fluorescence guidance as feedback. Other at-risk LNs could be sampled within the same regional basin. The pathological condition of all LNs was determined by histological examination.

[0029] Figures 8A through 8C illustrate the differentiation between metastatic and benign lymph nodes based on ICG patterns. (Figure 8A) NIRF imaging of a benign LN shows ICG fluorescence in the perinodal region. H&E histology and negative pan-cytokeratin staining were used to verify the absence of cancerous lesions. (Figure 8B) Micrometastatic LN shows some UPS5.3-ICG fluorescence in the LN core. (Figure 8C) Macrometastatic LN shows extensive ICG fluorescence patterns in the enlarged LN tissue. The ICG fluorescence patterns are associated with dense cytokeratin staining. Scale bars are 300 and 50 μm, respectively.

[0030] Figures 9A to 9C show the accumulation of UPS nanoparticles in large metastatic lymph nodes. (Figure 9A) H&E staining of axillary lymph nodes shows enlarged nodules. (Figure 9B) Immunohistochemical staining against cytokeratin shows the presence of cancerous lesions in the LN. (Figure 9C) Near-infrared fluorescence scanning of tissue sections shows UPS nanoparticles. 5.3 -ICG and UPS 6.1 -ICG accumulates in regions with pan-cytokeratin expression. At the same fluorescence scale as UPS5.3 and UPS6.1, UPS... 6.9 -ICG showed a much lower fluorescence intensity. A low-scale display showed UPS6.9 accumulation in the pan-cytokeratin-positive region. The scale bar is 300 μm.

[0031] Figures 10A and 10B Receiver operating characteristic (ROC) analysis of metastatic lymph node detection via UPS nanoparticles is shown. (Figure 10A) The ROC curves illustrate the sensitivity and specificity of detecting large metastatic LNs using the LICOR signal of the entire node. UPS5.3 has an AUC of 0.96, indicating high discriminative power. (Figure 10B) ROC analysis based on the median CR variable. UPS6.9 exhibits high discriminative power but has a lower ICG signal, as shown in Figure 6C. Detailed Implementation

[0032] The block copolymers of this invention comprise hydrophilic and hydrophobic polymer segments, wherein the hydrophobic polymer segments contain ionizable amine groups to provide pH sensitivity. The block copolymers form pH-activatable micelles (pHAM) nanoparticles based on the supramolecular self-assembly of these ionizable block copolymers. At higher pH values, the block copolymers assemble into micelles, while at lower pH values, the ionization of the amine groups in the hydrophobic polymer segments leads to micelle dissociation. Figure 1A and1B Micelle formation and its thermodynamic stability are driven by a delicate balance between hydrophobic and hydrophilic segments. Ionizable groups can act as tunable hydrophilic / hydrophobic blocks at different pH values, directly influencing the dynamic self-assembly of micelles. Micellarization can accelerate the ionization transition of amines in hydrophobic polymer segments, thus providing a rapid and ultrasensitive pH response.

[0033] I. Block copolymers

[0034] Some embodiments described herein depict micelle-based fluorescence imaging agents. In some embodiments, the micelles comprise a diblock copolymer of polyethylene glycol (PEG) and dibutylamino-substituted polymethyl methacrylate (PMMA) covalently conjugated with indocyanine green (ICG). In some embodiments, the PEG comprises a shell or surface stabilizing the micelles. In some embodiments, the micelle size is < 100 nm.

[0035] In some embodiments, the block copolymer of formula (I), or its pharmaceutically acceptable salt, solvate, or hydrate, is provided herein:

[0036]

[0037] in:

[0038] n is 113;

[0039] x is between 60 and 150;

[0040] y is between 0.5 and 1.5; and

[0041] R' is a halogen, -OH, or -C(O)OH.

[0042] In some embodiments, the block copolymer of formula (I) is a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate) copolymer indocyanine green conjugate. In some embodiments, the block copolymer of formula (I) is PEO113-b-(DBA60-150-r-ICG 0.5-1.5).

[0043] Many fluorescent dyes are known in the art. In some aspects of this disclosure, the fluorescent dye is a pH-insensitive fluorescent dye. In some embodiments, the fluorescent dye is paired with a fluorescence quencher to obtain an enhanced signal change upon activation. In some cases, the fluorescent dye is conjugated to the compound directly or through a linker moiety. In some embodiments, the fluorescent dye is conjugated to an amine of the compound via an amide bond. In some embodiments, the fluorescent dye is a coumarin, fluorescein, rhodamine, xanthan, BODIPY®, Alexa Fluor®, or anthocyanin dye. In some embodiments, the fluorescent dye is indocyanine green, AMCA-x, Marina Blue, PyMPO, Rhodamine Green™, tetramethylrhodamine, 5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine5, Cyanine5.5, and Cyanine7.5. In some embodiments, the fluorescent dye is indocyanine green (ICG). Indocyanine green (ICG) is commonly used in medical diagnostics.

[0044] In some implementations, the compound is not conjugated with a dye.

[0045] In some embodiments, the block copolymer of formula (I) is a compound. In some embodiments, the block copolymer of formula (I) is a diblock copolymer. In some embodiments, the block copolymer comprises hydrophilic polymer segments and hydrophobic polymer segments. In some embodiments, the hydrophilic polymer segments comprise poly(ethylene oxide) (PEO). In some embodiments, the size of the hydrophilic polymer segments is from about 2 kD to about 10 kD. In some embodiments, the size of the hydrophilic polymer segments is from about 3 kD to about 8 kD or from about 4 kD to about 6 kD. In some embodiments, the size of the hydrophilic polymer segments is about 5 kD.

[0046] In some implementations, the hydrophobic polymer segments comprise:

[0047]

[0048] Where x is approximately 20 to approximately 200. In some embodiments, x is approximately 60 to 150. In some embodiments, the hydrophilic polymer segment comprises dibutylamine.

[0049] In some embodiments, R' is a terminal capping group. In some embodiments, the terminal capping group is a product of atom transfer radical polymerization (ATRP). In some embodiments, R' is a halogen. In some embodiments, R' is Br. In some embodiments, R' is -OH. In some embodiments, R' is -COH. In some embodiments, R' is an acid. In some embodiments, R' is -C(O)OH. In some embodiments, R' is H.

[0050] In one aspect, the compounds described herein are in the form of pharmaceutically acceptable salts. Similarly, active metabolites of these compounds having the same type of activity are also included within the scope of this disclosure. Furthermore, the compounds described herein can exist in both non-solventized and solvated forms with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.). The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0051] II. Micelles and pH-responsive compositions

[0052] One or more block copolymers described herein can be used to form pH-responsive micelles and / or nanoparticles. In another aspect, micelles comprising one or more block copolymers of formula (I) are provided herein.

[0053] The size of the micelles is typically nanoscale (i.e., about 1 nm to 1 µm in diameter). In some embodiments, the size of the micelles is about 10 to about 200 nm. In some embodiments, the size of the micelles is about 20 to about 50 nm. In some embodiments, the size of the micelles is less than 100 nm in diameter. In some embodiments, the size of the micelles is less than 50 nm in diameter.

[0054] In another aspect, this document provides pH-responsive compositions comprising one or more formula (I) block copolymers. The pH-responsive compositions disclosed herein comprise one or more pH-responsive micelles and / or nanoparticles comprising formula (I) block copolymers. Each block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophobic polymer segment contains an ionizable amine group to provide pH sensitivity.

[0055] In some embodiments, the pH-responsive composition has a pH transition point and an emission spectrum. In some embodiments, the pH transition point is 4.8 to 5.5. In some embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the emission spectrum of the pH-responsive composition is 750 to 850 nm.

[0056] In another aspect, there is a developer comprising one or more block copolymers described herein.

[0057] How to use

[0058] In some implementations, the block copolymers and micelles described herein can be used to detect primary and metastatic tumor tissues (including lymph nodes), resulting in reduced tumor recurrence and reoperation rates.

[0059] In some embodiments, the block copolymers and micelles described herein are used in pH-responsive compositions or pH-responsive micelles. In some embodiments, the pH-responsive compositions are used for imaging physiological and / or pathological processes involving intracellular or extracellular pH changes.

[0060] Aerobic glycolysis (known as the Warburg effect) occurs in all solid cancers, in which cancer cells preferentially take up glucose and convert it into lactate. Lactate preferentially accumulates in the extracellular space due to monocarboxylic acid transporters. The resulting acidification of the extracellular space promotes extracellular matrix remodeling for further tumor invasion and metastasis.

[0061] Some embodiments described herein depict compounds that form micelles at physiological pH (7.35 to 7.45). In some embodiments, the compounds described herein are conjugated with ICG dyes. In some embodiments, the molecular weight of the micelles is greater than 2 × 10⁻⁶. 7 Dalton. In some embodiments, the molecular weight of the micelles is approximately 2.7 × 10⁻⁶. 7 Dalton. In some embodiments, the ICG dye is isolated within the micelle core at physiological pH (7.35 to 7.45) (e.g., during blood circulation), resulting in fluorescence quenching. In some embodiments, when the micelles encounter an acidic environment (e.g., tumor tissue), the micelles dissociate into molecules with an average molecular weight of about 3.7 × 10⁻⁶. 4 The micelles are individual compounds of Dalton, which allow activation of the fluorescence signal from the ICG dye, causing specific fluorescence emission in acidic environments (e.g., tumor tissue). In some embodiments, the micelles dissociate at pH levels below the pH transition point (e.g., the acidic state of the tumor microenvironment).

[0062] In some implementations, the fluorescence response is strong due to the abrupt phase transition that occurs between the hydrophobic-driven self-assembly of micelles (non-fluorescence-off state) and the co-dissociation of these micelles at a predetermined low pH (fluorescence-on state).

[0063] In some embodiments, the micelles described herein have a pH transition point and an emission spectrum. In some embodiments, the pH transition point is 4 to 8. In other embodiments, the pH transition point is 6 to 7.5. In still other embodiments, the pH transition point is 4.8 to 5.5. In some embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the pH transition point is about 5.3. In some embodiments, the pH transition point is about 5.4. In some embodiments, the pH transition point is about 5.5. In some embodiments, the emission spectrum is 400 to 850 nm. In some embodiments, the emission spectrum is 700 to 900 nm. In some embodiments, the emission spectrum is 750 to 850 nm.

[0064] In some cases, the pH-sensitive micelle compositions described herein have a narrow pH transition range. In some embodiments, the pH transition range (ΔpH) of the micelles described herein is... 10至90% The pH transition range of the micelles is less than 1 pH unit. In several embodiments, the pH transition range is less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, and less than about 0.1 pH units. In some embodiments, the pH transition range of the micelles is less than about 0.5 pH units. In some embodiments, the pH transition range is less than 0.25 pH units. In some embodiments, the pH transition range is less than 0.15 pH units.

[0065] The fluorescence activation ratio is a measure of the micelles' open / closed state. In some embodiments, the fluorescence activation ratio (i.e., the difference between associated and dissociated micelles) is greater than 75 times that of associated micelles. In some embodiments, the fluorescence activation ratio of the fluorescence signal is greater than 25. In some embodiments, the fluorescence activation ratio of the fluorescence signal is greater than 50.

[0066] In some implementations, pH-responsive micelles have a contrast ratio (CR). The contrast ratio (CR) is the amount of signal relative to the background signal and is calculated based on Equation 1:

[0067] (1).

[0068] In some embodiments, the pH-responsive micelles have high contrast. In some embodiments, the contrast ratio is greater than about 30, 40, 50, 60, 70, 80, or 90. In some embodiments, the contrast ratio is greater than 50. In some embodiments, the contrast ratio is greater than 60. In some embodiments, the contrast ratio is greater than 70.

[0069] In some implementations, the optical signal is a fluorescent signal.

[0070] In some embodiments, when imaging the intracellular environment, cells are brought into contact with micelles under conditions suitable for inducing micelle uptake. In some embodiments, the intracellular environment is part of the cell. In some embodiments, part of the cell is a lysosome or endosome. In some embodiments, the extracellular environment is the extracellular environment of tumor or vascular cells. In some embodiments, the extracellular environment is intravascular or extravascular. In some embodiments, imaging the pH of the tumor environment includes imaging one or more sentinel lymph nodes. In some embodiments, pH imaging of the tumor environment allows determination of tumor size and margins. In some embodiments, the cells may be cancer cells from metastatic tumors. In some embodiments, cancer cells are present in lymph nodes. Cancer cells in lymph nodes can be used to determine the presence of metastatic tumors that have spread beyond the original tumor.

[0071] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is cancer or carcinoma. Some exemplary cancers are selected from, but are not limited to, breast cancer, ovarian cancer, colon cancer, urinary tract cancer, bladder cancer, lung cancer, prostate cancer, brain cancer, head and neck cancer (NHSCC), colorectal cancer, and esophageal cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, or colorectal cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (NHSCC). In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is colorectal cancer.

[0072] certain terms

[0073] Unless otherwise stated, the following terms as used in this application have the definitions given below. The use of the term "comprising / including" and other forms is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0074] As used herein, “medicinal” means a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic; that is, the material is applied to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of the composition containing it.

[0075] The term "medicinal salt" refers to a form of therapeutic active agent consisting of a cationic form combined with a suitable anion, or, in some alternative embodiments, a form of therapeutic active agent consisting of an anionic form combined with a suitable cation. (See Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. SMBerge, LD Bighley, DC Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. PHStahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich:Wiley-VCH / VHCA, 2002.) Pharmaceutical salts are generally more readily and rapidly soluble in gastric and intestinal fluids compared to nonionic substances, and are therefore suitable for use in solid dosage forms. Furthermore, because its solubility is generally a function of pH, selective dissolution in one or more parts of the digestive tract is possible, and this ability can be manipulated as an aspect of delayed and sustained release behavior. Additionally, since salt-forming molecules can be balanced in a neutral state, channels across biological membranes can be modulated.

[0076] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound of formula (I) with an acid. In some embodiments, the compound of formula (A) (i.e., in its free base form) is basic and reacts with an organic or inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid. Organic acids include, but are not limited to, 1-hydroxy-2-naphthylcarboxylic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+), capricacid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (caprylic acid). Caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclosine, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactosidic acid, gentian acid, glucoheponic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, oxalic acid, palmitic acid, palmitic acid, phosphoric acid, propionic acid, pyroglutamic acid (-L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+L), thiocyanate, toluenesulfonic acid (p), and undecenoic acid.

[0077] In some embodiments, the compound of formula (A) is prepared as a chloride salt, sulfate salt, bromide salt, methanesulfonate, maleate, citrate, or phosphate.

[0078] In some embodiments, a pharmaceutically acceptable salt is obtained by reacting a compound of formula (A) with a base. In some embodiments, the compound of formula (A) is acidic and reacts with a base. In such cases, the acidic proton of the compound of formula (A) is replaced by a metal ion, such as lithium, sodium, potassium, magnesium, calcium, or aluminum ions. In some cases, the compounds described herein are coordinated with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, meglumine, N-methylglucosamine, dicyclohexylamine, and tris(hydroxymethyl)methylamine. In other cases, the compounds described herein form salts with amino acids, such as, but not limited to, arginine, lysine, etc. Acceptable inorganic bases for forming salts with compounds containing acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, etc. In some embodiments, the compounds provided herein are prepared as sodium salts, calcium salts, potassium salts, magnesium salts, melamine salts, N-methylglucosamine salts, or ammonium salts.

[0079] It should be understood that references to pharmaceutically acceptable salts include solvation forms. In some embodiments, the solvate comprises a stoichiometric or non-stoichiometric amount of solvent and is formed during a crystallization process using a pharmaceutically acceptable solvent such as water, ethanol, etc. A hydrate is formed when the solvent is water, or an alcohol is formed when the solvent is an alcohol. The solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. Additionally, the compounds provided herein are optionally present in both non-solventized and solvated forms.

[0080] The methods and formulations described herein include the use of N-oxides (if appropriate) or pharmaceutically acceptable salts of compounds having the structure of formula (A), and active metabolites of these compounds having the same type of activity.

[0081] In another embodiment, the compounds described herein are labeled with isotopes (e.g., with radioactive isotopes) or by other means, including but not limited to the use of chromophores or fluorescent portions, bioluminescent labeling, or chemiluminescent labeling.

[0082] The compounds described herein include isotopically labeled compounds that are identical to those described in the polyforms and structures presented herein, but in fact, one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that may be incorporated into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, iodine, and phosphorus, such as... 2 H, 3 H, 13 C 14 C 15 N、 18 O、 17 O、 35 S, 18 F, 36 Cl、 123 I, 124 I, 125 I, 131 I, 32 P and 33 P. In one aspect, the isotope-labeled compounds described herein, for example those incorporating a radioactive isotope (e.g. 3 H and 14 Those of type C) can be used for drug and / or substrate tissue distribution assays. In one aspect, substitution with isotopes (e.g., deuterium) offers certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements.

[0083] As used herein, the terms “pH-responsive system,” “pH-responsive composition,” “micelle,” “pH-responsive micelle,” “pH-sensitive micelle,” “pH-activated micelle,” and “pH-activated micelle (pHAM) nanoparticles” are used interchangeably to refer to micelles comprising one or more compounds that dissociate according to pH (e.g., above or below a certain pH). As a non-limiting example, at a given pH, the compound of formula (I) is substantially in micelle form. As the pH changes (e.g., decreases), the micelles begin to dissociate, and as the pH changes further (e.g., decreases further), the compound of formula (I) is substantially in a dissociated (non-micelle) form.

[0084] The term "pH transition range" as used in this article refers to the pH range at which micelles dissociate.

[0085] The “pH transition value” (pH) used in this article refers to the pH at which half of the micelles dissociate.

[0086] As used herein, "nanoprobe" refers to a pH-sensitive micelle containing an imaging labeled portion. In some embodiments, the labeled portion is a fluorescent dye. In some embodiments, the fluorescent dye is indocyanine green (ICG).

[0087] Unless otherwise stated, the following terms as used in this application have the definitions given below. The use of the term "comprising / including" and other forms is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0088] As used herein, the term "application" and its variations refer to methods that enable the delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral, duodenal, parenteral (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), topical, and rectal administration. Those skilled in the art are familiar with application techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0089] The terms “co-administration” as used in this article are intended to cover the administration of a chosen therapeutic agent to a single patient and are intended to include treatment regimens in which the agent is administered via the same or different routes of administration or at the same or different times.

[0090] As used herein, the term "effective amount" or "therapeutic effective amount" means an adequate quantity of an agent or compound administered that will, to a certain extent, reduce one or more symptoms of the disease or condition being treated. Results include a reduction and / or alleviation of the signs, symptoms, or cause of the disease, or any other desired alteration of the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant reduction in the symptoms of a disease. In any individual case, the appropriate "effective" amount may optionally be determined using techniques such as dose-escalation studies.

[0091] As used herein, the term "enhancement" or its variations mean to increase or prolong the potency or duration of a desired effect. Therefore, in relation to enhancing the effect of a therapeutic agent, the term "enhancement" refers to the ability to increase or prolong the effect of another therapeutic agent on the system in terms of potency or duration. As used herein, "enhancing effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system.

[0092] The term "object" or "patient" encompasses mammals. Some examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. In one respect, the mammal is the human being.

[0093] The term “treat” as used herein, or variations thereof, includes the relief, reduction or improvement of at least one symptom of a disease or condition, prevention of additional symptoms, suppression of a disease or condition, for example, prevention of the development of a disease or condition, relief of a disease or condition, remission of a disease or condition, relief of the condition caused by a disease or condition, or preventive and / or therapeutic cessation of the symptoms of a disease or condition.

[0094] While this disclosure supports the definitions of "alternative only" and "and / or," the use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated that it refers only to an alternative or that the alternatives are mutually exclusive. Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of the error of the apparatus or method used to determine the value. Under long-standing patent law, when used in conjunction with the word "comprising" in the claims or description, a noun not limited by a quantifier indicates one / more / a type unless specifically indicated.

[0095] Example

[0096] Compounds are prepared using standard organic chemistry techniques, such as those described in, for example, March's Advanced Organic Chemistry, 6.th Those published in Edition, John Wiley and Sons, Inc. Unless otherwise stated, standard methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology were used. Some abbreviations used herein are as follows:

[0097] Area under the AUC curve

[0098] BC breast cancer

[0099] CR contrast

[0100] HNSCC head and neck squamous cell carcinoma

[0101] hr hours

[0102] ICG-OSu: Indocyanine green succinimide ester

[0103] IV intravenous

[0104] kg

[0105] LN lymph nodes

[0106] mg mg

[0107] mL / ml

[0108] µg micrograms

[0109] NC not calculated

[0110] NIRF near-infrared fluorescence

[0111] ROC receiver operational characteristics

[0112] ROI (Region of Interest)

[0113] SLNB (sentinel lymph node biopsy)

[0114] UPS over-pH sensitivity

[0115] Example 1. Materials and Methods

[0116] Synthesis of block copolymers: The block copolymers of formula (I) described herein are synthesized using standard synthesis techniques or methods known in the art, in conjunction with the methods described in patent publications WO 2012 / 039741 and WO 2015 / 188157.

[0117] More specifically, copolymers UPS6.9 (PEPA-ICG), UPS6.1 (PDPA-ICG), and UPS5.3 (PDBA-ICG) were synthesized via atom transfer radical polymerization (ATRP) using polyethylene glycol (PEG)-bromine macroinitiators, respectively, with ethylpropyl aminoethyl methacrylate (EPA), dipropyl aminoethyl methacrylate (DPA), and dibutylaminoethyl methacrylate (DBA). ICG-sulfonyl-OSu (AAT Bioquest) was conjugated with a primary amine in methanol at a molar ratio of three fluorophores per polymer for 24 hours. Unconjugated ICG was removed by discontinuous percolation purification in methanol using a 10 kDa regenerated cellulose ultrafiltration disc (Amicon Bioseparations). ICG conjugation was quantified by UV-Vis spectroscopy in methanol at a polymer concentration of 10 μg / mL using a Shimadzu UV-1800.

[0118] Purified ICG copolymers in methanol were dispersed in ten times their volume of deionized water under acoustic treatment for micelle self-assembly. The micelles were purified by washing three times with deionized water in a 100 kDa centrifugal filter element (Amicon Bioseparations). The stock concentration of the micelles was maintained at 5.0 mg / mL. The micelle nanoparticles were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS. The micelles were isolated in phosphate-buffered saline (PBS) at discrete pH values ​​(polymer pKa ± 0.5 pH units). Figure 1D The sample was diluted to 0.1 mg / mL. Additionally, ICG fluorescence intensity as a function of pH was measured. The sample was imaged with LI-COR Pearl in an 800 nm channel at 85 μm resolution.

[0119] Animal studies: An orthotopic 4T1.2 BALB / cj model was used in eight-week-old mice. 1×10⁻⁶ BALB / cj broiler pads were implanted into the right fourth mammary fat pad. 6 4 to 5 weeks after primary tumor growth, UPS nanoparticles resulted in consistent, spontaneous LN metastasis to the ipsilateral axillary LN, and occasionally to the ipsilateral or contralateral cervical and groin LN. UPS nanoparticles were administered intravenously at 1.0 mg / kg in 0.9% saline to BALB / cj mice bearing 4T1.2.

[0120] Fluorescence imaging: Real-time fluorescence imaging was performed using an NIRF camera. Emission light was filtered with an 860±12 nm bandpass filter (ThorLabs) and focused with a 25 mm / F1.8 fixed-focus lens (Edmund Optics). The filtered emission wavelength was detected using a Blackfly SUSB3 camera (FLIR). Unless otherwise noted, images were recorded at 4 fps. Individual lung nuclei (LNs) were excised under the guidance of the fluorescence imaging system and stereotactic microscopy.

[0121] Quantitative NIRF imaging was performed using the LI-COR Pearl small animal imaging system. Image acquisition was performed in an 800 nm channel at 85 μm resolution. Quantization was performed in Image Studio software, with ROIs drawn using the freehand tool. The median pixel intensity of each ROI and the LI-COR signal were output. Fluorescent slides were scanned at 21 μm resolution using the LI-COR Odyssey imager. For ease of comparison, images were correlated with the same filters.

[0122] Histology: Following dissection, LN tissue was formalin-fixed, paraffin-embedded, and cut into three 5.0 μm sections per 500 μm section until tissue exhaustion. This resulted in three to four sets of three adjacent slides. The first slide was stained with hematoxylin and eosin using an automated staining system (Dakewe). The second slide was used for NIRF imaging. The third adjacent slide was used for pancytokeratin immunohistochemistry. Heat-induced antigen retrieval was performed at 110 psi for 17 minutes in Tris pH 9. The slides were blocked with mouse serum (Mouse on mouse blocking reagent, Vector Laboratories) for 1 hour. Incubation with anti-mouse pancytokeratin antibody (1:10 dilution; AE1 / AE3 clone; ThermoFisher) in 2.5% normal horse serum (Vector Laboratories) was performed at room temperature for 30 minutes. Detection of the primary antibody was performed at room temperature for 10 minutes using Immpress equine anti-mouse IgG polymer reagent (Mouse on mouse blocking reagent, Vector Laboratories). DAB substrate was added until color development. Benign lymph nodes (LNs) are classified as pan-cytokeratin-negative. Micrometastases are defined as pan-cytokeratin-positive clusters smaller than 2 mm in size. Macrometastatic LNs are those with pan-cytokeratin-positive clusters larger than 2 mm in size.

[0123] Immunohistochemical staining revealed the spatial colocalization between nanoparticles and LN macrophages. BALB / cj mice (8 weeks old) were intravenously injected with a 1.0 mg / kg nanoparticle solution in 0.9% saline. LNs were excised under the guidance of an NIRF camera system. LNs were embedded in OTC medium and frozen in liquid nitrogen. Frozen sections were sectioned at 12 μm intervals with 500 μm spacing. Sections were fixed in acetone at -20°C for 10 min, followed by drying at room temperature for 10 min. Next, sections were washed twice in 1×PBS for 5 min each time. Blocking was performed with normal goat serum for 1 hour. Blocking serum was aspirated and then incubated with primary antibodies: FITC anti-mouse CD169 (1:125; clone 3D6.112; batch B271952), PE anti-mouse F4 / 80 (1:50; clone BM8; batch B199614), and APC anti-mouse CD11b (1:50; clone M1 / 70; batch B279418). All antibodies were multiplexed in PBS containing 0.5% Tween and added to each tissue section. Incubation was performed overnight at 4°C. Sections were washed three times in PBS for 5 minutes each time. Cover slips were mounted with Diamond Mount and DAPI. The slides were imaged using a Keyence automated microscope.

[0124] Statistical analysis: The LI-COR signal and median CR value were grouped according to histological status. One-way ANOVA was used to analyze the statistical differences in means within each group (benign, micrometastasis, and macrometastasis). Tukey multiple comparisons were used to assess differences between the means of each group. The ROC Curve module under the "Wilson / Brown" method in Graph Pad Prism was used to compare differences between variables and groups. This statistic was maximized to determine thresholds for sensitivity and specificity.

[0125] Example 2. pH-sensitive nanoparticles exhibit synergistic fluorescence in response to ambient pH.

[0126] Three ultra-pH sensitive (UPS) block copolymers were synthesized. The copolymers exhibit discrete pH transitions to cover a range of pH responses (UPS5.3, UPS6.1, and UPS6.9; each subscript indicates apparent pK). a value)( Figure 1B (Table 1). In particular, the pK of the amphiphilic block copolymer UPS6.1 a It is 6.1. (This is higher than pK) a At the specified pH value, UPS6.1 self-assembled into micelles of 24.0 ± 2.1 nm. Figure 1C(Table 1). At pH values ​​below 6.1, protonation of the polymer chains led to micelle decomposition into 4.9 ± 1.2 nm monomers ( Figure 1C UPS5.3 (28.5 ± 1.5 nm) and UPS6.9 (23.4 ± 2.5 nm) also exhibited a significant pH-dependent transition from micelles to monomers (Table 1, Figure 2C). The comparable nanoparticle sizes (23 to 28 nm) and similar PEG lengths (5 kDa) among the micelle compositions are important for maintaining size and surface chemical consistency in LN targeting, thus enabling specific evaluation of the pH threshold when detecting LN transfer.

[0127] Table 1. Characterization of PEG-b-(PR-r-dye) nanoprobes.

[0128]

[0129] a Digital-based dimensions determined by dynamic light scattering. b Identification was performed using the LI-COR Pearl Imager via ICG fluorescence. c Determined by NaOH titration.

[0130] To report local pH values, each polymer was conjugated with indocyanine green (ICG), an FDA-approved fluorophore compatible with clinical near-infrared (NIRF) imaging systems. Each UPS-ICG nanoparticle shows a comparable copy of the dye for each polymer (Table 1, Figure 2A). However, in the micellar state at pH 7.4, homologous FRET-induced quenching eliminated the ICG fluorescence signal. Below pK a At pH 0.3, UPS micelles decompose into individual monomers and amplify fluorescence intensity by more than 50-fold over a pH span. Figure 1D (Table 2). USP nanoparticles demonstrate a binary encoding of the pH threshold by NIRF ( Figure 1D (2A and 2B, Table 2). This “digital” signal represents fluorescence activation as discrete values ​​at different pH thresholds (on = 1, off = 0).

[0131] Table 2. Measurement of the conjugation power and quantum yield of dye-conjugated copolymers.

[0132]

[0133] a Determined by a standard curve based on the UV-Vis spectrum of free ICG in methanol. b Identification was performed using a LI-COR PearlImager in 1×PBS via ICG fluorescence emission.

[0134] Example 3. Real-time systemic lymphatic localization in tumor-naïve mice to guide LN resection.

[0135] Each polymer nanoparticle formulation was administered intravenously to tumor-naïve BALB / cj mice to evaluate systemic lymph node localization. NIRF imaging revealed the dissected mice, clearly depicting the lymph nodes (LNs) in animals administered UPS5.3 and UPS6.1 (Figs. 3A and 3B). This depiction facilitated real-time image-guided resection of all superficial LNs. Quantitative imaging of excised tissues using LI-COR Pearl revealed comparable ICG signals from different anatomical groups of LNs. Median contrast ratio (CR) was calculated for all LN tissues (Equation 1):

[0136] (1)

[0137] LN fluorescence was amplified, and the median CR for all LN was 63.3 for UPS5.3 and 39.9 for UPS6.1 (Figure 3D). The median CR value for UPS6.9 was significantly lower than that for 10.7 (Figure 3D).

[0138] To explain the differences among the micelle compositions in LN targeting, pharmacokinetic studies were performed to evaluate fluorescence in tumor-initial BALB / cj plasma after intravenous injection (Fig. 4A). Compared to UPS5.3 and USP6.1, UPS6.9 was rapidly cleared from the blood (Fig. 4A). Furthermore, UPS6.9-ICG exhibited a low on / off ratio after plasma acidification, indicating that UPS6.9 decomposed 24 hours after intravenous injection (Fig. 4B). In normal mouse serum, all nanoparticles were stable for 24 hours during incubation, exhibiting high on / off ratios. The low on / off ratio of UPS6.9 is attributed to rapid clearance of the nanoprobe from the liver (Fig. 4C), leading to lower serum concentrations and an increased tendency for thermodynamic decomposition.

[0139] Biodistribution of micelles to lymph nodes (LNs) was shown to be a key parameter for distinguishing metastatic LNs. The blood half-life of UPS6.9 was lower than that of UPS6.1 and UPS5.3, as evidenced by increased accumulation in the livers of both tumor-bearing and tumor-initiated mice. To further investigate the role of biodistribution and circulation time in LN metastasis detection, additional circulation times were included at 6 and 72 hours following intravenous administration of UPS5.3 nanoparticles. Sinusoidal macrophages rapidly uptake the nanoparticles, as evidenced by a “halo” phenomenon in the LNs of the 6-hour group. However, longer circulation times did not allow for the differentiation of increased LN metastasis. Overall, the increased half-life of UPS5.3 enabled relatively better “capture and integration” of ICG fluorescence within the lymph node metastasis microenvironment.

[0140] Example 4. LN-residual macrophage internalization of UPS polymer micelles.

[0141] Although NIRF imaging depicts all superficial lymphocytes (LNs), the lymphotropic delivery mechanism remains unclear. Since reticuloendothelial systems containing phagocytes (e.g., liver, spleen) exhibit enhanced fluorescence intensity, theoretically, LN-resident macrophages are responsible for the uptake of UPS micelles, leading to amplification of the ICG fluorescence signal. Multiplex immunohistochemical (IHC) staining using a unique macrophage population and visualization of UPS nanoparticle uptake were employed. UPS5.3-ICG and UPS6.1-ICG fluorescence signals appeared in different regions of the LN (Figs. 5A and 5B). These regions showed significant overlap with LN-resident macrophages, particularly CD169. + / F4 / 80 + / CD11b + Macrophages colocalized with UPS5.3-ICG fluorescence. These cells shared the same biomarkers as LN-resident macrophages. Furthermore, ICG fluorescence did not correlate with F4 / 80 in adjacent tissues surrounding the LN. + Macrophage overlap supports the hypothesis of LN-specific delivery (Figures 5A and 5B), indicating that only LN-resident macrophages isolate UPS nanoparticles.

[0142] Example 5. Detection of metastatic LN in tumor-bearing mice.

[0143] The fluorescence intensity difference between metastatic and benign lung nuclei (LNs) was quantified using an syngeneic 4T1.2-BALB / cj mouse model. UPS5.3, UPS6.1, or UPS6.9 nanoparticles were administered intravenously at the same dose (1.0 mg / kg) for systemic detection of LN metastasis. After 24 hours of circulation, NIRF imaging of live mice using LICOR Pearl showed fluorescence emission within primary tumors but not in metastatic LNs (top left panel, Figures 6A-6C). In contrast, NIRF imaging of dissected mice showed accumulation in LNs in addition to primary tumors (top right panel, Figures 6A-6C). Animals administered UPS5.3 and UPS6.1 showed bright fluorescence signals in all superficial LNs (Figures 6A and 6B). Animals administered UPS6.9 showed micellar accumulation in enlarged LNs (Figure 6C). Real-time fluorescence imaging enabled guided resection of all LNs. Figures 7A and 7B Largely metastatic LNs typically differ from other LNs in fluorescence intensity, spatial pattern, and size, thus enabling precise excision of these LNs (Figure 7B).

[0144] The median contrast of all excised tissues was quantified (Equation 1). Additionally, the LI-COR signal was used to quantify the total fluorescence intensity from the region of interest (ROI). Each variable conveys unique information. Median CR evaluates the pixel-based median fluorescence intensity of the LN, while the LI-COR signal reports the total fluorescence intensity of the LN tissue. These two variables were evaluated in the statistical analysis of the grouped tissues. Histological examination of the LN allowed for tissue grouping based on pathology. LNs were classified as benign, micrometastatic (cancer lesions <2 mm), or macrometastatic (cancer lesions > 2 mm). The median CR and LI-COR signal values ​​were grouped accordingly (Figures 6D to F). Significant differences existed between the benign and macrometastatic groups (Figures 6D to F). However, no significant differences were shown between the benign and micrometastatic groups.

[0145] Example 6. Accumulation of UPS nanoparticles within metastatic LN cancer lesions.

[0146] In addition to differences in fluorescence intensity, distinct patterns of fluorescence signals were identified between benign and metastatic lung nuclei (LNs). Both benign LNs exhibited a “halo” of UPS5.3-ICG intensity in ex vivo imaging via real-time imaging (Figs. 7A, 7B, and 8A). Histological analysis confirmed the absence of pan-cytokeratin clusters in this LN subset (Fig. 8A). Furthermore, microscopic imaging revealed the accumulation of UPS nanoparticles at the tissue periphery of the LN (Fig. 8A). This pattern was also evident in animals treated with UPS6.1 and UPS6.9. The peripheral distribution of UPS5.3 nanoparticles in benign LNs co-localized with LN-resident macrophages in the LN sinusoid. These results are consistent with the fluorescence localization in tumor-naïve LNs (Fig. 4). However, in benign LNs from tumor-bearing mice, CD11b… + Macrophages showed greater motile activity in surrounding tissues compared to the same population in mice with tumor initiation.

[0147] Micrometastatic lung nuclei (LNs) exhibited fluorescence spectral characteristics. Fluorescence could be localized to the edges of the LN or show uniform fluorescence within small cancerous lesions. A mixed pattern of fluorescence localization at the edges and within pan-cytokeratin clusters was the most typical feature (Fig. 8B). In contrast, large metastatic LNs showed a broad pattern of fluorescence intensity (Fig. 8C). Microscopic analysis revealed that the ICG signal largely overlapped with anti-cytokeratin staining (Fig. 8C), indicating cancer-specific accumulation of UPS monomers. Similar results to those observed in the UPS6.1 group were observed. Furthermore, the fluorescence intensity of metastatic LN tissues from the UPS6.9 group was reduced compared to UPS6.1 and UPS5.3 (Fig. 9).

[0148] All three types of micelles showed accumulation in pan-cytokeratin-positive cancer lesions, resulting in detectable fluorescent signals. Quantification of fluorescence intensity suggests that the LICOR signal is a suitable indicator for differentiating LN metastases, particularly in the UPS5.3 group. Although LN-resident macrophage uptake of UPS nanoparticles leads to background fluorescence, the resulting fluorescence intensity can be quantitatively different from that of metastatic LNs. After delivery to the LN, macrophages internalize the micelles and amplify fluorescence within their acidic organelles. In contrast, metastatic LNs exhibit a broad fluorescence pattern throughout the LN cortex corresponding to the cancer lesion. This activation pattern can be detected by surgeons during resection. There is potential to further differentiate metastatic LNs by utilizing both fluorescence intensity and spatial localization.

[0149] Example 7. ROC differentiation between metastatic LN and benign LN.

[0150] Receiver operating characteristics (ROC) for the detection of macrometastatic LN were quantified (Table 3). Tissue quantification using size-dependent LICOR signals showed that UPS5.3 had high discriminative power against macrometastatic LN compared to benign LN (AUC = 0.96; sensitivity = 92.3% and specificity = 88.2%) (Figure 10A). It was also feasible to distinguish between benign and macrometastatic LN using the median CR for each polymer (Figure 10B). Data indicated a lack of discriminative power against micrometastases relative to benign LN at median CR or LICOR signals.

[0151] Table 3. Acceptor operation characteristics analysis of benign and microtransferable LNs of UPS nanoparticles.

[0152]

[0153] UPS = SuperpH Sensitivity; CR: Contrast Ratio; AUC = Area Under the Curve

[0154] While some preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used to implement the present disclosure. The appended claims are intended to define the scope of the present disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A block copolymer of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, or isotopic variant thereof: in: n is 113; x is between 60 and 150; y is between 0.5 and 1.5; and R' is a halogen, -COH, or -C(O)OH.

2. Micelles comprising one or more block copolymers according to claim 1.

3. A pH-responsive composition comprising the micelles of claim 2, wherein the micelles have a pH transition point and an emission spectrum.

4. The pH-responsive composition of claim 3, wherein the pH transition point is 6 to 7.

5.

5. The pH-responsive composition of claim 3, wherein the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.

5.

6. The pH-responsive composition according to any one of claims 3 to 5, wherein the emission spectrum is 700 to 850 nm.

7. The pH-responsive composition according to any one of claims 3 to 6, wherein the pH transition range (ΔpH) of the composition is... 10至90% (less than 1 pH unit) 8. The pH-responsive composition of claim 7, wherein the pH transition range is less than 0.25 pH units.

9. The pH-responsive composition of claim 7, wherein the pH transition range is less than 0.15 pH units.

10. The pH-responsive composition according to any one of claims 3 to 9, wherein the fluorescence activation ratio of the pH-responsive composition is greater than 25.

11. The pH-responsive composition according to any one of claims 3 to 10, wherein the fluorescence activation ratio of the pH-responsive composition is greater than 50.

12. The pH-responsive composition according to any one of claims 3 to 11, wherein the average contrast of the pH-responsive composition is greater than 50.

13. A developer comprising one or more block copolymers as described in claim 1.

14. The developer of claim 13, comprising a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate) copolymer indocyanine green conjugate.

15. A block copolymer comprising hydrophilic polymer segments and hydrophobic polymer segments, wherein the hydrophilic polymer segments comprise poly(ethylene oxide) (PEO) and the hydrophobic polymer segments comprise... Where x represents a total of approximately 20 to approximately 200.

16. The block copolymer of claim 15, wherein x is 60 to 150.

17. Methods for imaging the pH of the intracellular or extracellular environment, including: (a) Exposing the pH-responsive composition of claims 3 to 12 to the environment; as well as (b) Detect one or more light signals from the environment, wherein the detection of the light signals indicates that the micelles have reached their pH transition point and dissociated.

18. The method of claim 17, wherein the optical signal is a fluorescence signal.

19. The method of claim 17 or 18, wherein when imaging the intracellular environment, the cells are contacted with the pH-responsive composition under conditions suitable for inducing uptake of the pH-responsive composition.

20. The method of any one of claims 17 to 19, wherein the intracellular environment is part of the cell.

21. The method of any one of claims 17 to 19, wherein the extracellular environment is the extracellular environment of tumor or vascular cells.

22. The method of claim 21, wherein the extracellular environment is intravascular or extravascular.

23. The method of claim 21, wherein the tumor is cancer.

24. The method of claim 23, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer.

25. The method of claim 21, wherein the tumor is metastatic tumor cells.

26. The method of claim 25, wherein the metastatic tumor cells are located in lymph nodes.

Images