pH RESPONSIVE COMPOSITIONS AND USES THEREOF

Abstract

To provide pH responsive compounds, micelles, and compositions useful for the detection of primary and metastatic tumor tissues.SOLUTION: There are provided a block copolymer of Formula (I), a micelle comprising one or more kinds of the block copolymer, and a pH responsive composition comprising the micelle, wherein the micelle has a pH transition point and an emission spectrum.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] Statement Regarding Federally Sponsored Research This invention was made with United States government support under R01 EB 013149 and CA 192221 awarded by the National Institutes of Health. [Background technology]

[0003] background Approximately 1.7 million new cases of cancer are expected to be diagnosed and approximately 610,000 Americans are expected to die from cancer in 2019. Effective imaging agents are needed to detect primary and metastatic tumor tissue.

[0004] Treatment guidelines for all stages of solid tumors include surgical removal of at-risk or involved lymph nodes along with the surgical removal of the primary tumor. Despite biological and anatomical differences between these tumor types, the status of postoperative margins is one of the most important prognostic factors for local tumor control and, therefore, for the risk of disease recurrence or tumor metastasis. Surgical resection of solid tumors is a balance between oncological effectiveness and minimizing normal tissue resection, and is therefore correlated with mortality. This also applies to diagnostic and therapeutic lymph node dissections, which are often performed simultaneously with removal of the primary tumor. The presence or absence of lymph node metastasis is the most important determinant of survival for many solid tumors.

[0005] The adaptation of optical imaging strategies to intraoperative tissue imaging based on cellular imaging, natural autofluorescence, and Raman scattering is progressing rapidly. The potential of optical imaging includes the availability of camera systems that provide real-time feedback and a wide surgical field. One strategy to overcome the complexities resulting from the heterogeneity of cancer genotypes and histological phenotypes encountered during surgery is to target metabolic vulnerabilities universal to cancer. Aerobic glycolysis, known as the Warburg effect, in which cancer cells selectively take up glucose and convert it to lactate occurs in all solid tumors.

[0006] Thus, there remains a need to establish compositions and methods for determining the presence of cancer, particularly cancer metathesis in the lymphatic system. Summary of the Invention

[0007] overview The block copolymers presented herein exploit this universal pH difference between cancerous and normal tissues and, after cellular uptake, produce highly sensitive and specific fluorescent responses, thereby enabling the detection of tumor tissue, tumor margins, and metastatic tumors, including lymph nodes.

[0008] The compounds described herein are useful imaging agents for detecting primary tumor tissue and metastatic tumor tissue (including lymph nodes). Real-time fluorescence imaging during surgery assists surgeons in detecting metastatic lymph nodes or depicts tumor tissue in contrast to normal tissue, with the aim of achieving negative margins and complete tumor resection. Clinical benefits from improved surgical outcomes include, for example, reduced tumor recurrence and reoperation rates, avoidance of unnecessary surgery, and providing patient information about treatment plans.

[0009] In certain embodiments, provided herein is a block copolymer of formula (I): TIFF2025170333000002.tif78128In the formula, n is 113; x is 60 to 150; y is 0.5 to 1.5, and R' is halogen, -OH, or -C(O)OH.

[0010] In certain embodiments, provided herein are micelles comprising one or more block copolymers of Formula (I), or pharmaceutically acceptable salts, solvates, hydrates, or isotopic variants thereof.

[0011] In some embodiments, provided herein are pH-responsive compositions comprising micelles of a block copolymer of Formula (I), wherein the micelles have a pH transition point and an emission spectrum. In some embodiments, the pH transition point is between 4 and 8. In some embodiments, the pH transition point is between 6 and 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 less than 1 pH unit. 10~90% In some embodiments, the emission spectrum is 700-850 nm. In some embodiments, the pH transition range (ΔpH 10~90% In some embodiments, the emission spectrum is 700-850 nm. In some embodiments, the pH transition range (ΔpH 10~90% ).

[0012] In some embodiments, provided herein are methods for imaging the pH of an intracellular or extracellular environment, the methods comprising: (a) contacting the environment with a pH-responsive composition of the present disclosure; and (b) detecting one or more optical signals from the environment, wherein the detection of the optical signals indicates that the micelle has reached its pH transition point and dissociated. In some embodiments, the optical signals are fluorescent signals. In some embodiments, the intracellular environment is imaged, and the cell is contacted with the pH-responsive composition under conditions suitable to cause uptake of the pH-responsive composition. In some embodiments, the intracellular environment is part of a cell. In some embodiments, the extracellular environment is that of a tumor or a vascular cell. In some embodiments, the extracellular environment is intravascular or extravascular. In some embodiments, the tumor is of a cancer, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, ureter cancer, esophageal cancer, colon cancer, brain cancer, or skin cancer. In some embodiments, the tumor is a metastatic tumor cell. In some embodiments, the metastatic tumor cells are located in lymph nodes.

[0013] [The present invention 1001] A block copolymer of formula (I) TIFF2025170333000003.tif72128In formula, n is 113; x is between 60 and 150; y is between 0.5 and 1.5; and R' is halogen, -COH, or -C(O)OH; The block copolymer, or a pharmaceutically acceptable salt, solvate, hydrate, or isotopic variant thereof. [The present invention 1002] A micelle comprising one or more types of block copolymers of the present invention. [The present invention 1003] A pH-responsive composition comprising a micelle of the present invention 1002, the micelle having a pH transition point and an emission spectrum. [The present invention 1004] 1003. A pH-responsive composition of the present invention, wherein the pH transition point is 6 to 7.5. [The present invention 1005] 1003. The pH-responsive composition of the present invention, wherein the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. [The present invention 1006] 1006. The pH-responsive composition of any one of claims 1003 to 1005, wherein the emission spectrum is 700 to 850 nm. [The present invention 1007] pH transition range (ΔpH 10~90% 1007. The pH-responsive composition of any one of claims 1003 to 1006, comprising: [The present invention 1008] 1007. The pH-responsive composition of claim 10, wherein the pH transition range is less than 0.25 pH units. [The present invention 1009] 1007. The pH-responsive composition of claim 10, wherein the pH transition range is less than 0.15 pH units. [The present invention 1010] 1009. A pH-responsive composition according to any one of claims 1003 to 1009, having a fluorescence activation ratio of greater than 25. [The present invention 1011] The pH-responsive composition of any one of 1003 to 1010, having a fluorescence activation ratio of greater than 50. [The present invention 1012] 1003 to 1011, a pH-responsive composition of any one of the present inventions 1003 to 1011, having an average contrast ratio of greater than 50. [The present invention 1013] An imaging agent comprising one or more block copolymers of the present invention. [The present invention 1014] 1013. An imaging agent according to claim 1013, comprising a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate) copolymer indocyanine green conjugate. [The present invention 1015] A block copolymer comprising a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophilic polymer segment comprises poly(ethylene oxide) (PEO) and the hydrophobic polymer segment comprises TIFF2025170333000004.tif39128, wherein x is a total of about 20 to about 200. [The present invention 1016] 1015. A block copolymer of the present invention, wherein x is 60 to 150. [The present invention 1017] 1. A method for imaging pH of an intracellular or extracellular environment, comprising: (a) contacting any one of the pH-responsive compositions of the present invention 1003 to 1012 with the environment; and (b) detecting one or more optical signals from the environment, wherein detection of the optical signals indicates that the micelle has reached its pH transition point and dissociated. The method comprises: [The present invention 1018] 1017. The method of claim 1017, wherein said optical signal is a fluorescent signal. [The present invention 1019] The method of any one of claims 1017 to 1018, wherein when said intracellular environment is to be imaged, cells are contacted with said pH-responsive composition under conditions suitable to cause uptake of said pH-responsive composition. [The present invention 1020] 1019. The method of any one of claims 1017 to 1019, wherein the intracellular environment is part of a cell. [The present invention 1021] 1019. The method of any one of claims 1017 to 1019, wherein said extracellular environment is that of a tumor or that of vascular cells. [The present invention 1022] 1022. The method of claim 1021, wherein the extracellular environment is intravascular or extravascular. [The present invention 1023] The method of claim 1021, wherein the tumor is cancer. [The present invention 1024] The method of claim 1023, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, ureter cancer, esophageal cancer, colon cancer, brain cancer, or skin cancer. [The present invention 1025] 1022. The method of claim 1021, wherein the tumor is a metastatic tumor cell. [The present invention 1026] 1026. The method of claim 1025, wherein said metastatic tumor cells are located in lymph nodes. Other objects, features, and advantages of the compounds, methods, and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0014] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0015] [Figure 1]Figures 1A-1D show the dual fluorescence response of ultra-pH-sensitive (UPS) polymeric micelle probes. (Figure 1A) UPS micelles are self-assembled nanoparticles that disassemble into unimers in response to a threshold proton concentration. (Figure 1B) The amphiphilic block copolymer structure enables a cooperative pH response at a specific pKa. (Figure 1C) Dynamic light scattering shows distinct size populations of unimers (pH below the pKa) for USP6.1. (Figure 1D) Nonlinear amplification of fluorescence intensity indicates an ultra-pH-sensitive response to environmental pH signals. The inset tubes show near-infrared visualization of UPS5.3-ICG (top), UPS6.1-ICG (middle), and UPS6.9-ICG (bottom) as a function of pH. [Figure 2] Figures 2A-2C show the in vitro characterization of UPS-ICG nanoparticles. (Figure 2A) UPS-ICG nanoparticles absorb near-infrared light with a λmax of 788 nm. (Figure 2B) Raw mean fluorescence intensity of UPS-ICG nanoparticles measured in the 800 nm channel by LI-COR Pearl. (Figure 2C) Mean diameter of UPS-ICG nanoparticles measured by dynamic light scattering. [Figure 3] Figures 3A-3D show that whole-body near-infrared fluorescence imaging of dissected tumor-free BALB / cj mice enables real-time, image-guided resection of LNs. (Figure 3A) UPS5.3-ICG and (Figure 3B) UPS6.1-ICG delineate all superficial LNs, enabling image-guided resection. (Figure 3C) UPS6.9-ICG fluorescence is mostly sequestered in the liver. Image-guided resection of LNs is not possible. (Figure 3D) Median LN fluorescence intensity is normalized to that of skeletal muscle (Mu). Median CR of anatomical LN groups demonstrates dependence on the pKa of the polymeric micelles. UPS5.3 exhibits the greatest intensity within each anatomical LN group. [Figure 4]Figures 4A-4C show 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 indicate the "on" state of the nanoparticles. Plasma fluorescence was normalized to the fluorescence at time 0 to adjust for differences between UPS compositions. (Figure 4B) Fluorescence of acidified plasma was normalized to collected plasma, indicating the "on / off ratio." (Figure 4C) Ex vivo imaging of organs after 24 hours of circulating UPS nanoparticles. [Figure 5] Figures 5A-5C show the colocalization of UPS nanoparticles with macrophage subpopulations, demonstrating the uptake of micelles by lymph node-resident macrophages. (Figure 5A) UPS5.3-ICG colocalizes with CD169 (left), F4 / 80 (center), and CD11b (right), but the colocalization is restricted to the lymph node. White arrows indicate the colocalization of positive cells with ICG fluorescence. Light gray arrows indicate F4 / 80 cell staining in the absence of ICG fluorescence. (Figure 5B) The colocalization pattern of UPS6.1-ICG with macrophages is similar to that of UPS5.3-ICG. (Figure 5C) The fluorescence intensity of UPS6.9-ICG is much lower than that of UPS5.3-ICG and UPS6.1-ICG. All panels demonstrate phagocytosis of nanoparticles by macrophages in lymph nodes but not in surrounding tissues. The scale bar is 200 μm. [Figure 6]Figures 6A-6F show the detection of metastatic lymph nodes with histological verification. (Figure 6A) A representative example of a 4T1.2-bearing BALB / cj mouse administered UPS5.3-ICG demonstrates NIRF detection of the primary tumor (PT) using whole-body imaging, as well as delineation of benign LNs (Be), micrometastatic LNs (Mi), and macrometastatic LNs (Ma), enabling image-guided resection of inguinal LNs (In), axillary LNs (Ax), and cervical LNs (Cr). (Figure 6B) NIRF imaging of a mouse administered UPS6.1-ICG demonstrates delineation of the primary tumor and LNs, with benign LNs appearing almost as bright as metastatic LNs. (Figure 6C) UPS6.9-ICG accumulates in the liver (Li) with much higher intensity. While several macrometastatic LNs are delineated, many micrometastatic LNs are undetectable. (Figure 6D) UPS 5.3 signal and median CR of classified tissues show significant differences 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) UPS 6.1 signal and median CR of classified tissues show significant differences between macrometastatic and benign LNs, although the distribution of macrometastases is highly variable. (Figure 6F) UPS 6.9 signal and median CR of classified tissues show significant differences between macrometastatic and benign LNs. The signal variable is much less intense than UPS 5.3 and UPS 6.1. [Figure 7]Figures 7A and 7B show real-time resection of metastatic lymph nodes using NIR fluorescence guidance. (Figure 7A) 4T1.2-bearing BALB / cj mice 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 are depicted. (Figure 7B) LNs are visible in the anatomical region. Macrometastatic LNs exhibit increased fluorescence intensity, a unique spatial accumulation of fluorescence, and are larger than other LNs. These LNs are resected using NIR fluorescence guidance as feedback. Other at-risk LNs can be sampled in the same area. All LN lesions are confirmed by histological examination. [Figure 8] Figures 8A-8C show the differentiation of metastatic lymph nodes from benign lymph nodes based on ICG patterns. (Figure 8A) NIRF imaging of a benign LN shows ICG fluorescence at the outer edge of the lymph node. Histological analysis with H&E and negative pan-cytokeratin staining were used to verify the absence of cancer foci. (Figure 8B) Micrometastatic LNs show some UPS5.3-ICG fluorescence in the core of the LN. (Figure 8C) Macrometastatic LNs show a distinct, bright pattern of ICG fluorescence throughout the swollen LN tissue. The pattern of ICG fluorescence correlates with the intense staining for cytokeratin. Scale bars are 300 μm and 50 μm for the top and bottom, respectively. [Figure 9] Figures 9A-9C show the accumulation of UPS nanoparticles in macrometastatic lymph nodes. (Figure 9A) H&E staining of an axillary lymph node shows an enlarged lymph node. (Figure 9B) Anti-cytokeratin immunohistochemical staining reveals the presence of cancer foci in the LN. (Figure 9C) Near-infrared fluorescence scanning of tissue sections reveals that UPS5.3-ICG and UPS6.1-ICG accumulate in areas with pan-cytokeratin expression. UPS6.9-ICG exhibits much lower fluorescence intensity at the same fluorescence scale as UPS5.3 and UPS6.1. The lower scale shows the accumulation of UPS6.9 in pan-cytokeratin-positive areas. The scale bar is 300 µm. [Figure 10] Figures 10A and 10B show receiver operating characteristic (ROC) analysis of metastatic lymph node detection by UPS nanoparticles. (Figure 10A) ROC curve showing the sensitivity and specificity of macrometastatic LN detection using the LICOR signal of the whole lymph node. UPS 5.3 had an AUC of 0.96, indicating high discriminatory ability. (Figure 10B) ROC analysis based on the median values ​​of CR variables. UPS 6.9 has higher discriminatory ability, but it has a lower ICG signal, as shown in Figure 6C. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention The block copolymers of the present invention comprise hydrophilic and hydrophobic polymer segments, where the hydrophobic polymer segments contain ionizable amine groups that confer pH sensitivity. Based on the supramolecular self-assembly of these ionizable block copolymers, the block copolymers form pH-activatable micellar (pHAM) nanoparticles. At higher pH, the block copolymers assemble into micelles, while at lower pH, ionization of the amine groups in the hydrophobic polymer segments causes the micelles to dissociate (Figures 1A and 1B). Micelle formation and its thermodynamic stability are driven by a delicate balance between the hydrophobic and hydrophilic segments. The ionizable groups can act as tunable hydrophilic / hydrophobic blocks at different pH values, which can directly affect the dynamic self-assembly of the micelles. Micellization can abrupt the ionization transition of the amines in the hydrophobic polymer segments, resulting in a rapid and ultrasensitive pH response.

[0017] I. Block Copolymers Some embodiments provided herein describe micelle-based fluorescent imaging agents. In some embodiments, the micelles comprise a diblock copolymer of polyethylene glycol (PEG) and dibutylamino-substituted polymethyl methacrylate (PMMA) to which indocyanine green (ICG) is covalently conjugated. In some embodiments, the PEG constitutes the shell or surface of the stable micelles. In some embodiments, the micelles have a size of <100 nm.

[0018] In some embodiments, provided herein is a block copolymer of formula (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof: TIFF2025170333000005.tif78128In formula, n is 113; x is between 60 and 150; y is between 0.5 and 1.5; and R' is halogen, -OH, or -C(O)OH.

[0019] 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).

[0020] Numerous fluorescent dyes are known in the art. In some aspects of the present disclosure, the fluorescent dye is a pH-insensitive fluorescent dye. In some embodiments, the fluorescent dye is paired with a fluorescence quencher to increase the signal change upon activation. In some instances, the fluorescent dye is directly conjugated to the compound or conjugated via a linker moiety. In some embodiments, the fluorescent dye is conjugated to the amine of the compound via an amide bond. In some embodiments, the fluorescent dye is coumarin, fluorescein, rhodamine, xanthene, BODIPY®, Alexa Fluor®, or cyanine 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), which is often used in medical diagnostics.

[0021] In some embodiments, the compound is not conjugated to a dye.

[0022] 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 a hydrophilic polymer segment and a hydrophobic polymer segment. In some embodiments, the hydrophilic polymer segment comprises poly(ethylene oxide) (PEO). In some embodiments, the hydrophilic polymer segment is about 2 kD to about 10 kD in size. In some embodiments, the hydrophilic polymer segment is about 3 kD to about 8 kD in size, or about 4 kD to about 6 kD in size. In some embodiments, the hydrophilic polymer segment is about 5 kD in size.

[0023] In some embodiments, the hydrophobic polymer segment is TIFF2025170333000006.tif39128, wherein x is a total of about 20 to about 200. In some embodiments, x is about 60 to 150. In some embodiments, the hydrophilic polymer segment comprises dibutylamine.

[0024] In some embodiments, R' is an end group. In some embodiments, the end-capping group is the product of an atom transfer radical polymerization (ATRP) reaction. 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.

[0025] In one aspect, the compounds described herein are in the form of pharmaceutically acceptable salts.Similarly, the active metabolites of these compounds with the same activity are also included in the scope of the present disclosure.In addition, the compounds described herein can exist in solvated form with pharmaceutically acceptable solvents, such as water, ethanol, etc., and can also exist in unsolvated form.The solvated form of the compounds presented herein is also considered to be disclosed herein.

[0026] II. Micelles and pH-Responsive Compositions One or more of the block copolymers described herein can be used to form pH-responsive micelles and / or pH-responsive nanoparticles. In another aspect, provided herein are micelles comprising one or more block copolymers of formula (I).

[0027] The size of micelles is typically on the nanometer scale (i.e., about 1 nm to 1 μm in diameter). In some embodiments, micelles have a size of about 10 to about 200 nm. In some embodiments, micelles have a size of about 20 to about 50 nm. In some embodiments, micelles have a size of less than 100 nm in diameter. In some embodiments, micelles have a size of less than 50 nm in diameter.

[0028] In another aspect, provided herein is a pH-responsive composition comprising one or more block copolymers of Formula (I). The pH-responsive composition disclosed herein comprises one or more pH-responsive micelles and / or pH-responsive nanoparticles comprising the block copolymer of Formula (I). Each of the block copolymers comprises a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophobic polymer segment comprises an ionizable amine group that provides pH sensitivity.

[0029] 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 pH-responsive composition has an emission spectrum of 750 to 850 nm.

[0030] In another aspect, the imaging agent comprises one or more of the block copolymers described herein.

[0031] How to use In some embodiments, the block copolymers and micelles described herein are useful for detecting primary tumor tissue and metastatic tumor tissue (including lymph nodes), which results in reduced tumor recurrence and reduced reoperation rates.

[0032] In some embodiments, the block copolymers and micelles described herein are used as pH-responsive compositions or pH-responsive micelles. In some embodiments, the pH-responsive compositions are used to image physiological and / or pathological processes that involve changes in intracellular or extracellular pH.

[0033] Aerobic glycolysis, known as the Warburg effect, occurs in all solid tumors, where cancer cells selectively take up glucose and convert it to lactate. Lactate preferentially accumulates in the extracellular space via monocarboxylate transporters. The resulting acidification of the extracellular space promotes extracellular matrix remodeling for further tumor invasion and metastasis.

[0034] Some embodiments provided herein describe compounds that form micelles at physiological pH (7.35-7.45). In some embodiments, the compounds described herein are conjugated to an ICG dye. In some embodiments, the micelles form at 2×10 7 In some embodiments, the micelles have a molecular weight of about 2.7 x 10 Daltons. 7 In some embodiments, the ICG dye is sequestered within the micelle core at physiological pH (7.35-7.45) (e.g., in the blood circulation), which results in fluorescence quenching. In some embodiments, when the micelle encounters an acidic environment (e.g., tumor tissue), the micelle has a molecular weight of about 3.7 x 10 4 The micelles dissociate into individual compounds with an average molecular weight of 100 daltons, allowing the activation of the fluorescent signal from the ICG dye, resulting in a state in which the ICG dye specifically fluoresces in an acidic environment (e.g., tumor tissue). In some embodiments, the micelles dissociate at a pH below the pH transition point (e.g., in the acidic conditions of the tumor microenvironment).

[0035] In some embodiments, the fluorescent response is strong due to a sharp phase transition between the hydrophobic-driven self-assembly of micelles (non-fluorescent off state) and the cooperative dissociation of these micelles (fluorescent on state) at a predetermined low pH.

[0036] 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 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.

[0037] In some instances, the pH-sensitive micelle compositions described herein have a narrow pH transition range. In some embodiments, the micelles described herein have a pH transition range (ΔpH 10~90% In various embodiments, the micelles have a pH transition range of less than about 0.9 pH units, less than about 0.8 pH units, less than about 0.7 pH units, less than about 0.6 pH units, less than about 0.5 pH units, less than about 0.4 pH units, less than about 0.3 pH units, less than about 0.2 pH units, or less than about 0.1 pH units. In some embodiments, the micelles have a pH transition range of 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.

[0038] The fluorescence activation ratio is a measure of the on / off state of the micelles. 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 fluorescent signal has a fluorescence activation ratio greater than 25. In some embodiments, the fluorescent signal has a fluorescence activation ratio greater than 50.

[0039] In some embodiments, the pH-responsive micelles have an average contrast ratio (CR), which is the amount of signal relative to the background signal and is calculated based on the following formula 1: TIFF2025170333000007.tif8128.

[0040] In some embodiments, the pH-responsive micelles have a high contrast ratio. 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.

[0041] In some embodiments, the optical signal is a fluorescent signal.

[0042] In some embodiments, when the intracellular environment is imaged, the cell is contacted with the micelle under conditions suitable for causing uptake of the micelle. In some embodiments, the intracellular environment is a portion of a cell. In some embodiments, the portion of a cell is a lysosome or an endosome. In some embodiments, the extracellular environment is that of a tumor or a vascular cell. 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, imaging the pH of the tumor environment allows for determining the size and margins of the tumor. In some embodiments, the cell can be a cancer cell from a metastatic tumor. In some embodiments, the cancer cell is present in a lymph node. Cancer cells in lymph nodes can be used to determine the presence of metastatic tumors that have spread beyond the original tumor.

[0043] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a cancer or carcinoma. Exemplary cancers are selected from, but not limited to, breast cancer, ovarian cancer, colon cancer, urinary tract cancer, bladder cancer, lung cancer, prostate cancer, brain cancer, NHSCC, colorectal cancer, and esophageal cancer. In some embodiments, the cancer is breast cancer, NHSCC, esophageal cancer, or colorectal cancer. In some embodiments, the cancer is breast cancer, NHSCC, lung cancer, ovarian cancer, prostate cancer, bladder cancer, ureter cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is NHSCC. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is colorectal cancer.

[0044] Specific Terms Unless otherwise stated, the following terms used in this application have the definitions set forth below. The use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0045] "Pharmaceutically acceptable," as used herein, refers to a substance, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compound and is relatively non-toxic, i.e., it may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0046] The term "pharmaceutically acceptable salt" refers to a form of a therapeutically active agent that consists of the cationic form of the therapeutically active agent combined with a suitable anion, or in another embodiment, the anionic form of the therapeutically active agent combined with a suitable cation. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. SM Berge, LD Bighley, DC Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. PH Stahl and CG Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich:Wiley-VCH / VHCA, 2002. Pharmaceutical salts are typically more soluble and more rapidly dissolving in gastric and intestinal fluids than non-ionic species and are therefore useful as solid dosage forms. Furthermore, their solubility is often pH-related, allowing them to be selectively dissolved in one part of the gastrointestinal tract or another, and this ability can be manipulated as an aspect of delayed- and sustained-release behavior. Also, salt-forming molecules can equilibrate with the neutral form, allowing for tunable passage through biological membranes.

[0047] In some embodiments, a pharmaceutically acceptable salt is obtained by reacting a compound of Formula (I) with an acid. In some embodiments, a compound of Formula (A) (i.e., in free base form) is basic and is reacted 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-naphthoic 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 (+); capric acid (decanoic acid); caproic acid (hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecylsulfuric acid; ethane-1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; glycerophosphoric acid; 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; pamoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (- L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+ L); thiocyanic acid; toluenesulfonic acid (p); and undecylenic acid.

[0048] In some embodiments, the compound of formula (A) is prepared as a chloride, sulfate, bromide, mesylate, maleate, citrate, or phosphate salt.

[0049] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound of Formula (A) with a base. In some embodiments, a compound of Formula (A) is acidic and reacts with a base. In such situations, the acidic proton of a compound of Formula (A) is replaced by a metal ion, for example, a lithium ion, a sodium ion, a potassium ion, a magnesium ion, a calcium ion, or an aluminum ion. 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-methylglucamine, dicyclohexylamine, and tris(hydroxymethyl)methylamine. In other cases, the compounds described herein form salts with amino acids, such as, but not limited to, arginine and lysine. Acceptable inorganic bases used to form 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, and the like. In some embodiments, the compounds provided herein are prepared as sodium, calcium, potassium, magnesium, melamine, N-methylglucamine, or ammonium salts.

[0050] It should be understood that a reference to a pharmaceutically acceptable salt includes solvent addition forms. In some embodiments, solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are formed during the crystallization process with a pharmaceutically acceptable solvent, such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein optionally exist in both solvated and unsolvated forms.

[0051] The methods and formulations described herein include the use of N-oxides (where appropriate) or pharmaceutically acceptable salts of compounds having the structure of Formula (A), as well as the use of active metabolites of these compounds that have the same type of activity.

[0052] In another embodiment, the compounds described herein are isotopically labeled (e.g., with a radioisotope) or by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0053] The compounds described herein include isotopically labeled compounds, which are identical to those listed in the various formulas and structures presented herein, but in fact, one or more atoms are replaced by atoms with atomic masses or mass numbers different from those usually found in nature.Examples of isotopes that can be incorporated into the compounds 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 In one aspect, the isotopically labeled compounds described herein, such as 3 H and 14Incorporation of radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium offers several therapeutic advantages due to increased metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0054] As used herein, the terms "pH-responsive system," "pH-responsive composition," "micelle," "pH-responsive micelle," "pH-sensitive micelle," "pH-activatable micelle," and "pH-activatable micelle (pHAM) nanoparticle" are used interchangeably herein to refer to micelles containing one or more compounds that dissociate in a pH-dependent manner (e.g., above or below a certain pH). As a non-limiting example, at a certain pH, the compound of formula (I) is substantially in a micellar form. As the pH changes (e.g., decreases), the micelles begin to dissociate, and as the pH changes (e.g., decreases further), the compound of formula (I) exists substantially in a dissociated (non-micellar) form.

[0055] As used herein, "pH transition region" refers to the pH range at which micelles dissociate.

[0056] As used herein, "pH transition value" (pH) refers to the pH at which half of the micelles dissociate.

[0057] The term "nanoprobe" is used herein to refer to a pH-sensitive micelle containing a labeling moiety for imaging. In some embodiments, the labeling moiety is a fluorescent dye. In some embodiments, the fluorescent dye is indocyanine green (ICG).

[0058] Unless otherwise stated, the following terms used in this application have the definitions set forth below. The use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0059] The terms "administer," "administering," "administration," and the like, as used herein, refer to techniques that can be used to enable delivery of a compound or composition to a desired site of biological action. These techniques include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with the administration techniques available for the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0060] "Co-administration" and like terms, as used herein, are intended to encompass the administration of more than one selected therapeutic agent to a single patient and include treatment regimens in which the multiple therapeutic agents are administered by the same or different routes of administration, or at the same time or at different times.

[0061] The term "effective amount" or "therapeutically effective amount," as used herein, refers to a sufficient amount of an administered agent or compound to relieve to some extent one or more symptoms of the disease or disorder being treated.The results include reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system.For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound as disclosed herein that is required to provide a clinically significant reduction in disease symptoms.The appropriate "effective" amount in any individual case can be optionally determined using techniques such as, for example, a dose escalation study.

[0062] The terms "enhance" or "enhancing," as used herein, means to increase or prolong either in potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents in a system. An "enhancing-effective amount," as used herein, refers to an amount adequate to enhance the effect of another therapeutic agent in a desired system.

[0063] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates, e.g., chimpanzees and other apes and monkey species; livestock animals, e.g., cows, horses, sheep, goats, pigs; domesticated animals, e.g., rabbits, dogs, and cats; laboratory animals, including rodents, e.g., rats, mice, and guinea pigs; and the like. In one aspect, the mammal is a human.

[0064] The terms "treat," "treating," or "treatment," as used herein, include alleviating, attenuating, or ameliorating at least one symptom of a disease or disorder, preventing additional symptoms, inhibiting a disease or disorder, e.g., arresting the progression of a disease or disorder, relieving a disease or disorder, causing regression of a disease or disorder, alleviating a condition caused by a disease or disorder, or arresting the symptoms of a disease or disorder, either prophylactically and / or therapeutically.

[0065] The use of the term "or" in the claims is used to mean "and / or" unless it is expressly specified to refer to alternatives only or the alternatives are mutually exclusive, but this disclosure supports that definition referring to alternatives only and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value. In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or this specification, mean one, or more than one, unless specifically specified. [Example]

[0066] The compounds can be prepared using standard organic chemistry techniques, e.g., March's Advanced Organic Chemistry, 6 th The preparation is carried out using techniques described in, for example, the American Chemical Society, John Wiley and Sons, Inc. Unless otherwise specified, conventional techniques in mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are utilized. Some abbreviations used herein are as follows: Area under the AUC curve BC Breast Cancer CR contrast ratio HNSCC Head and neck squamous cell carcinoma hr time ICG-OSu: Indocyanine green succinimide ester IV (intravenous) kg kilogram LN lymph node mg milligram mL milliliter μg microgram NC Not calculated NIRF Near-infrared Fluorescence ROC Receiver Operating Characteristic ROI Region of Interest SLNB Sentinel lymph node biopsy UPS ultra pH sensitive

[0067] Example 1. Materials and Methods Synthesis of Block Copolymers: The block copolymers of formula (I) described herein are synthesized using standard synthetic techniques or methods known in the art in combination with methods described in patent publications numbered WO 2012 / 039741 and WO 2015 / 188157.

[0068] More specifically, ethylpropylaminoethyl methacrylate (EPA), dipropylaminoethyl methacrylate (DPA), and dibutylaminoethyl methacrylate (DBA) were used to synthesize copolymers UPS 6.9 (PEPA-ICG), UPS 6.1 (PDPA-ICG), and UPS 5.3 (PDBA-ICG) from polyethylene glycol (PEG) bromide macroinitiators by atom transfer radical polymerization (ATRP). ICG-sulfo-OSu (AAT Bioquest) was conjugated to primary amines at a molar ratio of three fluorophores per polymer in methanol for 24 hours. Unconjugated ICG was removed by discontinuous diafiltration in methanol using 10 kDa regenerated cellulose ultrafiltration disks (Amicon Bioseparations). Conjugation of ICG is quantified by UV-visible spectroscopy using a Shimadzu UV-1800 at a polymer concentration of 10 μg / mL in methanol.

[0069] To self-assemble micelles, purified ICG copolymer in methanol was dispersed in 10 volumes of deionized water by sonication. The micelles were purified by washing three times with deionized water in a 100 kDa centrifugal filter unit (Amicon Bioseparations). The stock concentration of micelles was maintained at 5.0 mg / mL. The micellar nanoparticles were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS. Micelles were diluted to 0.1 mg / mL in phosphate-buffered saline (PBS) at different pHs (± 0.5 pH units from the pKa of the polymer, Figure 1D). Additionally, ICG fluorescence intensity was measured as a function of pH. The samples were imaged using a LI-COR Pearl at 800 nm channel and 85 μm resolution.

[0070] Animal Study: An orthotopic 4T1.2 BALB / cj model was utilized in 8-week-old mice. 1×10 6 Transplantation of these cells resulted in consistent spontaneous LN metastasis to the ipsilateral axillary LN and occasional metastasis to the ipsilateral cervical and inguinal LNs after 4–5 weeks of primary tumor growth. UPS nanoparticles were administered intravenously at 1.0 mg / kg in 0.9% saline into 4T1.2-bearing BALB / cj mice.

[0071] Fluorescence imaging: Real-time fluorescence imaging was performed using a NIRF camera. The emitted light was filtered using an 860 ± 12 nm bandpass filter (ThorLabs) and focused using a 25 mm / F1.8 fixed focal length lens (Edmund Optics). The filtered emission wavelength was detected using a Blackfly S USB3 camera (FLIR). Images were recorded at 4 fps unless otherwise specified. Individual LNs were excised under the guidance of the fluorescence imaging system and microscope using stereotaxic techniques.

[0072] Quantitative NIRF imaging was performed using a LI-COR Pearl Small Animal Imaging System. Image acquisition was performed in the 800 nm channel at 85 μm resolution. Quantification was performed in Image Studio software, with ROIs drawn using the freehand tool. Median pixel intensity and LI-COR signal were exported for each ROI. Fluorescent slides were scanned at 21 μm resolution using a LI-COR Odyssey imager. Images were linked to the same filter for ease of comparison.

[0073] Histological Analysis: After dissection, LN tissues were formalin-fixed, paraffin-embedded, and sectioned into three 5.0 μm slices at 500 μm intervals until the tissue was exhausted. This resulted in three to four groups of three adjacent slides. The first slide was stained with hematoxylin and eosin using an automated stainer (Dakewe). The second slide was used for NIRF imaging. The third adjacent slide was used for pan-cytokeratin immunohistochemical analysis. Heat-induced antigen retrieval was achieved in Tris, pH 9, at 110 psi for 17 minutes. Slides were blocked for 1 hour with mouse serum (Mouse on Mouse Blocking Reagent, Vector Laboratories). Incubation with anti-mouse pan-cytokeratin antibody (1:10 dilution; AE1 / AE3 clone; ThermoFisher) in 2.5% normal horse serum (Vector Laboratories) was performed at room temperature for 30 minutes. Primary antibody detection was performed using Immpress horse anti-mouse IgG polymer reagent (mouse-on-mouse blocking reagent, Vector Laboratories) for 10 minutes at room temperature. DAB substrate was added until color development occurred. Benign LNs were classified as pan-cytokeratin negative. Micrometastases were defined as pan-cytokeratin-positive clusters less than 2 mm in size. Macrometastatic LNs were those with pan-cytokeratin-positive clusters greater than 2 mm in size.

[0074] Immunohistochemical staining allowed visualization of the spatial colocalization of 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 a NIRF camera system. LNs were embedded in OTC medium and frozen using liquid nitrogen. The frozen sections were sectioned at 12 μm intervals at 500 μm intervals. The sections were fixed in -20°C acetone for 10 minutes, followed by drying at room temperature for 10 minutes. The sections were then washed twice in 1x PBS for 5 minutes each. Blocking was performed with normal goat serum for 1 hour. After aspiration of the blocking serum, the sections were incubated with the following primary antibodies: FITC anti-mouse CD169 (1:125; clone 3D6.112; lot number B271952), PE anti-mouse F4 / 80 (1:50; clone BM8; lot number B199614), and APC anti-mouse CD11b (1:50; clone M1 / 70; lot number 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. Coverslips were mounted using Diamond Mount with DAPI. Slides were imaged using a Keyence automated microscope.

[0075] Statistical analysis: LI-COR signal and median CR values ​​were grouped according to histological analysis status. Each group (benign, micrometastasis, and macrometastasis) was analyzed for statistical differences in means using one-way analysis of variance. Differences between the means of each group were assessed using Tukey's multiple comparisons. To compare discrimination between variables and groups, the "ROC Curve" module using the "Wilson / Brown" method was used in GraphPad Prism. This statistic was maximized to determine thresholds for sensitivity and specificity.

[0076] Example 2. pH-sensitive nanoparticles exhibit a coordinated fluorescence response to environmental pH Three ultra-pH-sensitive (UPS) block copolymers were synthesized. These copolymers had different pH transitions (UPS5.3, UPS6.1, and UPS6.9; the subscripts indicate the apparent pK a values ​​are shown) (Figure 1B, Table 1). Specifically, the amphiphilic block copolymer UPS6.1 has a pK a pK a At pH values ​​above 6.1, UPS 6.1 self-assembles into micelles measuring 24.0 ± 2.1 nm (Figure 1C, Table 1). Below a pH of 6.1, protonation of the polymer chains causes disassembly of the micelles into unimers measuring 4.9 ± 1.2 nm (Figure 1C). UPS 5.3 (28.5 ± 1.5 nm) and UPS 6.9 (23.4 ± 2.5 nm) also exhibit a sharp pH-dependent transition from micelles to unimers (Table 1, Figure 2C). Similar nanoparticle sizes (23–28 nm) and identical PEG lengths (5 kDa) across micelle compositions are important for maintaining consistent size and surface chemistry in LN targeting, allowing for specific assessment of the pH threshold for detecting LN transition.

[0077] Table 1. Characterization of PEG-b-(PR-r-dye) nanoprobes TIFF2025170333000008.tif25138 a Size based on number determined by dynamic light scattering. b Determined by ICG fluorescence using a LI-COR Pearl Imager. c Determined by NaOH titration.

[0078] To determine the local pH, each polymer was conjugated with indocyanine green (ICG), a fluorophore that is FDA-approved and compatible with clinical near-infrared (NIRF) imaging systems. Each of the UPS-ICG nanoparticles exhibits a similar number of dyes per polymer (Table 1, Figure 2A). However, in the micellar state at pH 7.4, homoFRET-induced quenching negates the ICG fluorescence signal. pK a At pHs below 0.3, UPS micelles disassemble into individual unimers, amplifying fluorescence intensity by more than 50-fold within a pH range of 0.3 (Figure 1D, Table 2). USP nanoparticles exhibit dual encoding of pH thresholds by NIRF (Figures 1D, 2A, and 2B, Table 2). This "digital" signal represents fluorescence activation as different values ​​(on = 1, off = 0) at different pH thresholds.

[0079] Table 2. Measurement of conjugation efficiency and quantum yield of dye-conjugated copolymers. TIFF2025170333000009.tif33128 a Determined by a standard curve based on UV-visible spectroscopy of free ICG in methanol. b Determined by ICG fluorescence in 1x PBS using a LI-COR Pearl Imager.

[0080] Example 3. Real-time whole-body lymphatic mapping in tumor-free mice guides LN resection To evaluate systemic lymphatic mapping, each of the polymer nanoparticle formulations was intravenously administered to tumor-free BALB / cj mice. NIRF imaging visualized dissected mice and clearly delineated the LNs in animals treated with UPS 5.3 and 6.1 (Figures 3A and 3B). This delineation facilitated real-time, image-guided resection of all superficial LNs. Quantitative ex vivo imaging of excised tissues using LI-COR Pearl demonstrated similar ICG signals from different anatomical groups of LNs. Median contrast ratios (CR) were calculated for all LN tissues (Equation 1): TIFF2025170333000010.tif8128. LN fluorescence was amplified (Figure 3D), with median CR values ​​for all LNs of 63.3 for UPS 5.3 and 39.9 for UPS 6.1. The median CR value for UPS 6.9 was significantly lower at 10.7 (Figure 3D).

[0081] To clarify the reasons for the differences in LN targeting between the micelle compositions, a pharmacokinetic study was conducted to evaluate fluorescence in the plasma of tumor-free BALB / cj mice after intravenous injection (Figure 4A). Compared with UPS5.3 and USP6.1, UPS6.9 cleared from the blood more rapidly (Figure 4A). In addition, UPS6.9-ICG had a low on / off ratio after plasma acidification, indicating that UPS6.9 decomposed within 24 hours after intravenous injection (Figure 4B). All nanoparticles with high on / off ratios were stable for more than 24 hours during incubation in normal mouse serum. The low on / off ratio of UPS6.9 was attributed to the rapid clearance of the nanoprobe in the liver (Figure 4C), which lowered serum concentrations and increased the thermodynamic tendency for degradation.

[0082] The biodistribution of micelles to LNs is considered a critical parameter for the differentiation of metastatic LNs. UPS 6.9 has a shorter blood half-life than UPS 6.1 and UPS 5.3, as indicated by its increased accumulation in the livers of both tumor-bearing and non-tumor-bearing mice. To further investigate the effect of biodistribution and circulation time on the detection of LN metastasis, additional circulation times of 6 and 72 hours after intravenous administration of UPS 5.3 nanoparticles were included. Given the presence of a "halo" phenomenon in LNs from the 6-hour group, sinusoidal macrophages rapidly uptake the nanoparticles. However, longer circulation times do not appear to enhance the differentiation of LN metastases. Overall, the increased half-life of UPS 5.3 allows for better "capture and incorporation" of ICG fluorescence within the lymph node metastasis microenvironment.

[0083] Example 4. LN-resident macrophages internalize UPS polymeric micelles NIRF imaging depicts all superficial LNs, but the lymphotropic delivery mechanism remains unclear. Because the reticuloendothelial system (e.g., liver, spleen) containing phagocytes has increased fluorescence intensity, it is theorized that LN-resident macrophages play a role in the uptake of UPS micelles, resulting in the amplification of the ICG fluorescence signal. Multiplexed immunohistochemistry (IHC) staining of different macrophage populations was utilized to visualize UPS nanoparticle uptake. Fluorescent signals of UPS5.3-ICG and UPS6.1-ICG appeared in distinct regions in LNs (Figures 5A and 5B). These regions specifically showed significant overlap with LN-resident macrophages and were associated with CD169 expression. + / F4 / 80 + / CD11b + Macrophages colocalize with UPS5.3-ICG fluorescence. These cells share the same biomarkers as LN-resident macrophages. In addition, ICG fluorescence is also associated with F4 / 80 in the adjacent tissue around the LN. +There was no overlap with macrophages, supporting the assumption of LN-specific delivery (FIGS. 5A and 5B), indicating that only LN-resident macrophages sequester UPS nanoparticles.

[0084] Example 5. Detection of metastatic LNs in tumor-bearing mice The difference in fluorescence intensity of metastatic LNs compared with benign LNs was quantified using a syngenic 4T1.2-BALB / cj mouse model. For systemic detection of LN metastases, UPS 5.3, UPS 6.1, or UPS 6.9 nanoparticles were intravenously administered at the same dose (1.0 mg / kg). After 24 hours of circulation, NIRF imaging of live mice using LICOR Pearl demonstrated fluorescence emission within the primary tumor but not within metastatic LNs (Figures 6A-6C, top, left panels). In contrast, NIRF imaging of dissected mice showed accumulation in LNs in addition to the primary tumor (Figures 6A-6C, top, right panels). Animals treated with UPS 5.3 and UPS 6.1 showed strong fluorescence signals in all superficial LNs (Figures 6A and 6B). Animals treated with UPS 6.9 showed accumulation of micelles in swollen LNs (Figure 6C). Real-time fluorescence imaging enabled guided resection of all LNs (Figures 7A and 7B). Macrometastatic LNs often differed from other LNs in fluorescence intensity, spatial pattern, and size, allowing precise resection of these LNs (Figure 7B).

[0085] The median contrast ratio was quantified for all excised tissues (Equation 1). Additionally, the LI-COR signal was used to quantify the total fluorescence intensity from the region of interest (ROI). Each variable provides different information. The median CR evaluates the pixel-based median fluorescence intensity of the LN, while the LI-COR signal conveys the summed fluorescence intensity of the LN tissue. Both variables were evaluated in statistical analyses of the grouped tissues. Histological examination of the LNs allowed for the tissue grouping based on lesion. LNs were classified as either benign, micrometastatic (cancerous lesions < 2 mm), or macrometastatic (cancerous lesions > 2 mm). The median CR and LI-COR signal values ​​were grouped accordingly (Figures 5D–5F). There was a significant difference between the benign and macrometastatic groups (Figures 5D–5F). However, no micellar groups showed significant differences between benign and micrometastatic lesions.

[0086] Example 6. UPS nanoparticles accumulate within cancerous lesions of metastatic LNs In addition to differences in fluorescence intensity, distinct patterns of fluorescence signal were identified between benign and macrometastatic LNs. Benign LNs exhibit a "halo" of UPS5.3-ICG intensity by both real-time and ex vivo imaging (Figures 7A, 7B, and 8A). Histological analysis confirmed the absence of pan-cytokeratin clusters in this LN subset (Figure 8A). Furthermore, microscopic imaging confirmed the accumulation of UPS nanoparticles at the rim of LN tissue (Figure 8A). This pattern was also evident in animals treated with UPS6.1 and UPS6.9. In benign LNs, the distribution of UPS5.3 nanoparticles at the periphery colocalized with resident macrophages in the LN sinusoids. These results are consistent with the localization of fluorescence in non-tumor-bearing LNs (Figure 4). However, in benign LNs from tumor-bearing mice, CD11b + Macrophages appear to be more motile within the surrounding tissue compared to the same population in non-tumor-bearing mice.

[0087] Micrometastatic LNs exhibit a spectrum of fluorescence signatures. Fluorescence may be localized at the edge of the LN or may show uniform fluorescence throughout the small cancerous lesion. A mixed pattern of fluorescence localized at the edge and within pan-cytokeratin clusters is the most typical signature (Figure 8B). In contrast, macrometastatic LNs exhibit a distinct and bright pattern of fluorescence intensity (Figure 8C). Microscopic analysis showed that the ICG signal nearly overlapped with anti-cytokeratin staining (Figure 8C), indicating the accumulation of UPS unimers, which are specific to cancer. Similar results were observed in the UPS 6.1-treated group. Furthermore, the fluorescence intensity of metastatic LN tissue from the UPS 6.9 group was reduced compared to UPS 6.1 and UPS 5.3 (Figure 9).

[0088] All three types of micelles showed accumulation in pan-cytokeratin-positive cancer foci, resulting in detectable fluorescent signals. Quantification of fluorescence intensity revealed that the LICOR signal is an appropriate metric for achieving differentiation of LN metastases, particularly in the UPS5.3 group. Although uptake of UPS nanoparticles by LN-resident macrophages contributes to background fluorescence, the resulting fluorescence intensity is quantifiably distinct from metastatic LNs. Upon delivery to the LN, macrophages internalize the micelles and amplify the fluorescence within their acidic organelles. Conversely, metastatic LNs exhibit a distinct, bright pattern of fluorescence throughout the LN cortex, corresponding to the cancer foci. This pattern of activation may be detectable by surgeons during resection. It may be possible to utilize both the intensity and spatial localization of fluorescence to achieve better differentiation of metastatic LNs.

[0089] Example 7. ROC discrimination of metastatic LNs from benign LNs The receiver operating characteristics (ROC) for macrometastatic LN detection were quantified (Table 3). Quantification of tissues with size-dependent LI-COR signal reveals that UPS5.3 has high discriminatory power for macrometastatic LNs versus benign LNs (AUC = 0.96; sensitivity = 92.3%, and specificity = 88.2%) (Figure 10A). Discrimination of benign LNs from macrometastatic LNs using the median CR for each polymer is also feasible (Figure 10B). The data show a lack of discrimination of micrometastatic LNs versus benign LNs using either the median CR or the LICOR signal.

[0090] Table 3. Receiver operating characteristic analysis of benign LNs vs. micrometastatic LNs for UPS nanoparticles. TIFF2025170333000011.tif69152UPS = ultra pH sensitivity; CR: contrast ratio; AUC = area under the curve.

[0091] While 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. Numerous changes, modifications, and substitutions will now 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 may be utilized in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

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

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

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

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

5.

5. 4. 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. pH transition range (ΔpH 10～90% 7. The pH-responsive composition according to claim 3, wherein the pH-responsive composition comprises:

8. 8. The pH-responsive composition of claim 7, wherein the pH transition range is less than 0.25 pH units.

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

10. 10. The pH-responsive composition of claim 3, having a fluorescence activation ratio of greater than 25.

11. 11. The pH-responsive composition of claim 3, having a fluorescence activation ratio of greater than 50.

12. 12. The pH-responsive composition of claim 3, having an average contrast ratio of greater than 50.

13. 10. An imaging agent comprising one or more block copolymers according to claim 1.

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

15. A block copolymer comprising a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophilic polymer segment comprises poly(ethylene oxide) (PEO) and the hydrophobic polymer segment comprises wherein the total number of x's is from about 20 to about 200.

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

17. 1. A method for imaging pH of an intracellular or extracellular environment, comprising: (a) contacting the environment with the pH-responsive composition of any one of claims 3 to 12; and (b) detecting one or more optical signals from the environment, wherein detection of the optical signals indicates that the micelle has reached its pH transition point and dissociated. The method comprises:

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

19. 19. The method of claim 17 or 18, wherein when the intracellular environment is to be imaged, the cell is contacted with the pH-responsive composition under conditions suitable to cause uptake of the pH-responsive composition.

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

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

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

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

24. 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, ureter cancer, esophageal cancer, colon cancer, brain cancer, or skin cancer.

25. 22. The method of claim 21, wherein the tumor is a metastatic tumor cell.

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

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