Biodegradable ultra-pH sensitive polymers

Abstract

The present disclosure relates to degradable polymers containing pH-sensitive hydrophobic and hydrophilic segments. In some aspects, the polymers have a backbone that can form pH-sensitive micelles and undergo in vivo degradation. In some aspects, the present disclosure also provides methods of using these degradable polymers for drug delivery.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 865,187, filed June 22, 2019, the entire contents of which are incorporated herein by reference.

[0002] This invention was made with Government support under Grant Nos. R01 CA216839 and U01 CA218422 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 1. Field The present disclosure relates generally to the fields of molecular and cell biology, drug delivery, and nanotechnology. More specifically, the present disclosure relates to degradable polymers that can be used to deliver therapeutic agents. [Background technology]

[0004] 2. Description of Related Technology Stimuli-responsive materials have rapidly advanced into a wide range of medical applications in recent years (Blum et al., 2015; Reineke, 2016; Torchilin, 2014; Yang et al., 2016). Various pH-sensitive polymers and nanoparticles have been designed and studied for tumor imaging and drug delivery applications (Moitra et al., 2013; Moitra et al., 2014; Zhang et al., 2015). For many biological applications, the differences between organelle pH at different maturation states, such as early endosomes versus late endosomes, or between tumor microenvironment pH and normal tissue pH, are small, including pH differences as small as less than 1 pH unit (Casey et al., 2009; Gerweck and Seetharaman, 1996), and thus are difficult for traditional small-molecule or polymeric pH sensors to discern. Recently, a series of ultra-pH-sensitive (UPS) copolymers with binary responses to environmental pH have been developed. Ionizable residues such as tertiary amines with different hydrophobic substituents were introduced onto the backbone of poly(methyl methacrylate) (PMMA) polymers. It was theorized that hydrophobic micellization contributes to sharp pH response (e.g., fluorescence on / off activation within 0.25 pH units). The resulting transistor-like nanoparticles showed improved accuracy in multiple biological applications, including tumor imaging and surgery (Zhou et al., 2011a; Zhou et al., 2011b; Wang et al., 2013; Zhao et al., 2016), imaging and perturbation of endocytic organelles and lysosomal catabolism (Wang et al., 2015; Wang et al., 2017), and nanovaccines for cancer immunotherapy (Wang et al., 2016; Luo et al., 2017).

[0005] As mentioned above, current UPS copolymers are synthesized with a non-degradable PMMA polymer backbone, which hampers the ability of these polymers to be excreted from the body. For certain medical applications, such as cancer surgery, the frequency of use (i.e., a single injection prior to surgery) and low toxicity of the imaging agent (e.g., indocyanine green) make the PMMA design safe for human use. However, in other applications requiring repeated injections, such as drug or gene delivery, the PMMA design may result in excessive material accumulation in the body, limiting the safety of long-term clinical use of these UPS copolymers.

[0006] Thus, there remains a need to develop and manufacture polymeric systems that can be used to deliver therapeutic agents that can be degraded in vivo. Summary of the Invention

[0007] overview In some aspects, the present disclosure provides a compound of the formula: or a pharma- ceutically acceptable salt thereof; During the ceremony: R1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 20 to 200; z is an integer from 0 to 200; where either y or z monomers are randomly distributed in the polymer; X1, X2, X1′, and X2′ are each independently O or NR a where: R a is an alkyl (C≦6) or substituted alkyl (C≦6) and; R2 and R2' are each independently hydrogen, alkyl, (C≦8) , or substituted alkyl (C≦8) and; R3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X3 and X3' are each independently O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L and L′ are each independently a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1, Y2, Y1', and Y2' are each independently an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 or Y1′ and Y2′ taken together are alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0008] In some embodiments, the polymer is further defined as the following, or a pharma- ceutically acceptable salt thereof: TIFF0007676331000002.tif32128 where: R1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 20 to 200; X1 and X2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1 and Y2 each independently represent an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 taken together are an alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0009] In some embodiments, the polymer is further defined as the following, or a pharma- ceutically acceptable salt thereof: TIFF0007676331000003.tif32128 where: R1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; x is an integer from 10 to 200; y is an integer from 20 to 200; X1 and X2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1 and Y2 each independently represent an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 taken together are an alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0010] In some embodiments, the polymer is further defined as the following, or a pharma- ceutically acceptable salt thereof: TIFF0007676331000004.tif32128 where: R1 is hydrogen, alkyl (C≦8), substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 20 to 200; X1 and X2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1 and Y2 each independently represent an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 taken together are an alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0011] In some embodiments, the polymer is further defined as the following, or a pharma- ceutically acceptable salt thereof: TIFF0007676331000005.tif32128 where: R1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 20 to 200; R2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; L is a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1 and Y2 each independently represent an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 taken together are an alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0012] In some embodiments, the polymer is further defined as the following, or a pharma- ceutically acceptable salt thereof: TIFF0007676331000006.tif32128 where: x is an integer from 10 to 200; y is an integer from 20 to 200; R2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; L is a group of the formula: -X4-S(O) n -X5- During the ceremony: n is 0, 1, or 2; and X4 and X5 each independently represent an alkanediyl group. (C≦8) or substituted alkanediyl (C≦8) and Y1 and Y2 each independently represent an alkyl group. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y1 and Y2 taken together are an alkanediyl; (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group.

[0013] In some embodiments, p is 1. In some embodiments, q is 1. In some embodiments, m is 1, 2, or 3, e.g., 2. In some embodiments, X1 is O. In some embodiments, X2 is O. In some embodiments, X3 is O.

[0014] In some embodiments, R1 is alkyl (C≦8) or substituted alkyl (C≦8) In some embodiments, R is alkyl. (C≦8) In some embodiments, R3 is hydrogen. In some embodiments, R2 is alkyl. (C≦8) or substituted alkyl (C≦8) In some embodiments, R2 is alkyl (C≦8) , for example methyl.

[0015] In some embodiments, X4 of L is alkanediyl. (C≦6) or substituted alkanediyl (C≦6) In some embodiments, X4 of L is alkanediyl. (C≦6) In some embodiments, X5 of L is alkanediyl. (C≦6) or substituted alkanediyl (C≦6) In some embodiments, X5 of L is alkanediyl. (C≦6)In some embodiments, n is 0.

[0016] In some embodiments, Y is alkyl. (C≦12) or substituted alkyl (C≦12) In some embodiments, Y is alkyl. (C2~12) or substituted alkyl (C2~12) In some embodiments, Y is alkyl. (C2~12) In some embodiments, Y1 is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, Y2 is alkyl. (C≦12) or substituted alkyl (C≦12) In some embodiments, Y2 is alkyl. (C2~12) or substituted alkyl (C2~12) In some embodiments, Y2 is alkyl. (C2~12) In some embodiments, Y2 is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, Y1 and Y2 taken together are alkanediyl. (C≦12) or substituted alkanediyl (C≦12) In some embodiments, Y and Y taken together are alkanediyl. (C≦8) or substituted alkanediyl (C≦8) In some embodiments, Y and Y taken together are alkanediyl. (C≦8) For example, Y1 and Y2 taken together are -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2-.

[0017] In some embodiments, the polymer comprises: TIFF0007676331000007.tif99136TIFF0007676331000008.tif150137, or a pharma- ceutically acceptable salt thereof.

[0018] In some embodiments, x is an integer from 40 to 160, for example, an integer from 80 to 150. In some embodiments, y is an integer from 40 to 180, for example, an integer from 80 to 150.

[0019] In yet another aspect, the present disclosure provides a micelle comprising a plurality of the polymers described herein.

[0020] In still yet another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (A) a polymer as described herein, and (B) Therapeutic Agent wherein the polymer encapsulates a therapeutic agent.

[0021] In some embodiments, the polymer forms a micelle. In some embodiments, the micelle completely encapsulates the therapeutic agent. In some embodiments, the therapeutic agent is an agent that affects the immune system, such as a cytokine or an immune system regulator. In some embodiments, the cytokine is IL-1β, IL-2, IL-12, or IL-15. In other embodiments, the immune system regulator is cGAMP or type 1 interferon. In other embodiments, the therapeutic agent is an antigen, such as an anti-cancer antigen. In some embodiments, the anti-cancer antigen is E7 peptide.

[0022] In some embodiments, the composition is formulated as a pharmaceutical composition and further comprises an excipient.In some embodiments, the pharmaceutical composition is formulated for administration orally, intraadiposely, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, by liposome, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, by cream, by lipid composition, by catheter, by lavage, by continuous infusion, by injection, by inhalation, by injection, by local delivery, or by local perfusion.In some embodiments, the pharmaceutical composition is formulated for administration by injection, for example, formulated for intraarterial, intramuscular, intraperitoneal, intratumoral, or intravenous administration. In some embodiments, the excipient is a vehicle, such as an aqueous solution suitable for injection.

[0023] In another aspect, the present disclosure provides a method for treating a disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the composition described herein, wherein the therapeutic agent is sufficient to treat the disease or disorder.In some embodiments, the disease or disorder is cancer.In some embodiments, the therapeutic agent can modulate the immune system to target cancer.In some embodiments, the therapeutic agent generates an immune response against one or more cancer cells.

[0024] As used herein, "pH-responsive micelles," "pH-sensitive micelles," "pH-activatable micelles," and "pH-activatable micellar (pHAM) nanoparticles" are used interchangeably herein to refer to micelles comprising one or more block copolymers that dissociate depending on the pH (e.g., above or below a certain pH). As a non-limiting example, at a certain pH, the block copolymers are substantially in micellar form. As the pH is changed (e.g., decreased), the micelles begin to dissociate, and as the pH is changed further (e.g., decreased further), the block copolymers are present in a substantially dissociated (non-micellar) form.

[0025] As used herein, "pH transition range" refers to the pH range above which micelles dissociate.

[0026] As used herein, the term "pH transition value" (pH t ) indicates the pH at which half of the micelles dissociate.

[0027] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0028] The terms "comprise" (and any form of including, such as "comprises" and "comprising"), "have" (and any form of having, such as "has" and "having"), "contain" (and any form of including, such as "contains" and "containing"), and "include" (and any form of including, such as "includes" and "including") are open-ended linking verbs. As a result, a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more recited steps or elements has those recited steps or elements, but is not limited to having only those steps or elements; it may have (i.e., cover) elements or steps that are not recited. Similarly, an element of a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more recited features has those features, but is not limited to having only those features; it may have features that are not recited.

[0029] Any embodiment of any of the methods, compositions, kits, and systems of the present invention may consist of or consist essentially of the recited steps and / or features, rather than comprising / comprising / containing / having the recited steps and / or features. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" may be used in place of any of the open-ended linking verbs set forth above in order to modify the scope of a given claim from that which would otherwise use an open-ended linking verb.

[0030] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to only alternatives or unless the alternatives are mutually exclusive, although the disclosure supports a definition that refers only to alternatives and "and / or."

[0031] 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 employed to determine the value.

[0032] In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in a claim or specification, mean one or more, unless specifically stated otherwise.

[0033] [The present invention 1001] formula: TIFF0007676331000009.tif33128 or a pharma- ceutically acceptable salt thereof, During the ceremony: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 20 to 200; z is an integer from 0 to 200; where either y or z monomers are randomly distributed in the polymer; X 1 、X 2 、X 1 ′, and X 2 ' are each independently O or NR a where: R a is an alkyl (C≦6) or substituted alkyl (C≦6) and; R 2 and R 2 ' are each independently hydrogen, alkyl, (C≦8) , or substituted alkyl (C≦8) and; R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X 3 and X 3 ' are each independently O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L and L′ are each independently a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 、Y 2 、Y 1 ′, and Y 2 Each ' is independently alkyl. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 Or Y 1 ′ and Y 2 ' are combined and form alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, polymer. [The present invention 1002] below: TIFF0007676331000010.tif32128 or a pharma- ceutically acceptable salt thereof; where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 20 to 200; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of the present invention 1001. [The present invention 1003] below: TIFF0007676331000011.tif32128 or a pharma- ceutically acceptable salt thereof; where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; x is an integer from 10 to 200; y is an integer from 20 to 200; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of the present invention 1001 or 1002. [The present invention 1004] below: TIFF0007676331000012.tif32128 or a pharma- ceutically acceptable salt thereof; where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 20 to 200; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl(C≦12) or a substituted form of either group, A polymer according to any one of 1001 to 1003 of the present invention. [The present invention 1005] below: TIFF0007676331000013.tif32128 or a pharma- ceutically acceptable salt thereof; where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 20 to 200; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, A polymer according to any one of 1001 to 1004 of the present invention. [The present invention 1006] below: TIFF0007676331000014.tif32128 or a pharma- ceutically acceptable salt thereof; where: x is an integer from 10 to 200; y is an integer from 20 to 200; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and; L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, Any of the polymers 1001 to 1005 of the present invention. [The present invention 1007] The polymer of the present invention 1001 or 1002, wherein p is 1. [The present invention 1008] The polymer of any one of 1001, 1002, or 1007, wherein q is 1. [The present invention 1009] A polymer according to any one of claims 1001 to 1003, 1007, and 1008, wherein m is 1, 2, or 3. [The present invention 1010] The polymer of the present invention 1009, wherein m is 2. [The present invention 1011] X 1 The polymer of any one of 1001 to 1004 and 1007 to 1010 of the present invention, wherein [The present invention 1012] X 2 The polymer according to any one of 1001 to 1004 and 1007 to 1011 of the present invention, wherein [The present invention 1013] X 3 The polymer according to any one of 1001 to 1004 and 1007 to 1012 of the present invention, wherein [The present invention 1014] R 1 Is alkyl (C≦8) or substituted alkyl (C≦8) The polymer of any one of the present inventions Nos. 1001 to 1005 and 1007 to 1013, [The present invention 1015] R 1 Is alkyl (C≦8) The polymer of the present invention 1014. [The present invention 1016] R 1 The polymer of the present invention, wherein is methyl. [The present invention 1017] R 3 The polymer according to any one of claims 1001 to 1005 and 1007 to 1016, wherein [The present invention 1018] R 2 Is alkyl (C≦8) or substituted alkyl (C≦8) The polymer of any one of claims 1001 to 1017, [The present invention 1019] R 2 Is alkyl (C≦8) The polymer of the present invention 1018. [The present invention 1020] R 2 The polymer of the present invention, wherein is methyl. [The present invention 1021] L's X 4 But, alkanediyl (C≦6) or substituted alkanediyl (C≦6) The polymer of any one of 1001 to 1020 according to the present invention, [The present invention 1022] L's X 4 But, alkanediyl (C≦6) The polymer of the present invention 1021. [The present invention 1023] L X 4 But -CH 2 CH 2 The polymer of the present invention 1022, [The present invention 1024] L's X 5 But, alkanediyl (C≦6) or substituted alkanediyl (C≦6) The polymer of any one of claims 1001 to 1023 of the present invention. [The present invention 1025] L's X 5 But, alkanediyl (C≦6) The polymer of the present invention 1024. [The present invention 1026] L X 5 But -CH 2 CH 2 The polymer of the present invention 1025, [The present invention 1027] The polymer of any one of 1001 to 1026 according to the present invention, wherein n is 0. [The present invention 1028] Y 1 But alkyl (C≦12) or substituted alkyl (C≦12) The polymer of any one of claims 1001 to 1027, [The present invention 1029] Y 1 But alkyl (C2~12) or substituted alkyl (C2~12) The polymer of the present invention 1028. [The present invention 1030] Y 1 Is alkyl (C2~12) The polymer of the present invention 1028 or 1029, [The present invention 1031] Y 1 The polymer of any one of claims 1028 to 1030, wherein is methyl, ethyl, n-propyl, or n-butyl. [The present invention 1032] Y 2 But alkyl (C≦12) or substituted alkyl (C≦12) The polymer of any one of Nos. 1001 to 1031 according to the present invention. [The present invention 1033] Y 2 But alkyl (C2~12) or substituted alkyl (C2~12) The polymer of the present invention 1032. [The present invention 1034] Y 2 Is alkyl (C2~12) The polymer of the present invention 1032 or 1033, [The present invention 1035] Y 2 The polymer of any one of claims 1032 to 1034, wherein is methyl, ethyl, n-propyl, or n-butyl. [The present invention 1036] Y 1 and Y 2 But together, alkanediyl (C≦12) or substituted alkanediyl (C≦12) The polymer of any one of claims 1001 to 1027, [The present invention 1037] Y 1 and Y 2 But together, alkanediyl (C≦8) or substituted alkanediyl (C≦8) The polymer of the present invention 1036. [The present invention 1038] Y 1 and Y 2 But together, alkanediyl (C≦8) The polymer of the present invention 1037. [The present invention 1039] Y 1 and Y 2 are combined and -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 The polymer of the present invention 1037, [The present invention 1040] below: TIFF0007676331000015.tif99136TIFF0007676331000016.tif150137 or a pharma- ceutically acceptable salt thereof. [The present invention 1041] The polymer according to any one of 1001 to 1040 of the present invention, wherein x is an integer of 40 to 160. [The present invention 1042] The polymer of the present invention, wherein x is an integer of 80 to 150. [The present invention 1043] The polymer of any one of 1001 to 1042 according to the present invention, wherein y is an integer of 40 to 180. [The present invention 1044] The polymer of the present invention, wherein y is an integer of 80 to 150. [The present invention 1045] A micelle comprising a plurality of polymers according to any one of claims 1001 to 1044 of the present invention. [The present invention 1046] (A) any one of the polymers 1001 to 1044 of the present invention, and (B) Therapeutic Agent wherein the polymer encapsulates the therapeutic agent. [The present invention 1047] The composition of claim 1046, wherein the polymer forms micelles. [The present invention 1048] The composition of claim 1046 or 1047, wherein the micelles completely encapsulate the therapeutic agent. [The present invention 1049] The composition of any of claims 1046 to 1048, wherein the therapeutic agent is an agent that affects the immune system. [The present invention 1050] The composition of the present invention 1049, wherein the therapeutic agent is a cytokine or an immune system modulating agent. [The present invention 1051] The composition of the present invention 1050, wherein the cytokine is IL-1β, IL-2, IL-12, or IL-15. [The present invention 1052] The composition of the present invention 1050, wherein the immune system modulating agent is cGAMP or type 1 interferon. [The present invention 1053] The composition of the present invention 1049, wherein the therapeutic agent is an antigen. [The present invention 1054] The composition of the present invention, wherein the antigen is an anti-cancer antigen. [The present invention 1055] The composition of the present invention, wherein the anti-cancer antigen is an E7 peptide. [The present invention 1056] The composition of any one of claims 1046 to 1055, which is formulated as a pharmaceutical composition and further comprises an excipient. [The present invention 1057] The composition of the present invention 1056, wherein the pharmaceutical composition is formulated for administration orally, intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, by liposomes, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, by cream, by lipid composition, by catheter, by lavage, by continuous infusion, by injection, by inhalation, by injection, by local delivery, or by local perfusion. [The present invention 1058] The composition of claim 1057, wherein the pharmaceutical composition is formulated for administration by injection. [The present invention 1059] The composition of the present invention 1057, wherein the pharmaceutical composition is formulated for intra-arterial, intramuscular, intraperitoneal, intratumoral, or intravenous administration. [The present invention 1060] The composition of any one of claims 1056 to 1059, wherein the excipient is a vehicle. [The present invention 1061] The composition of the present invention 1060, wherein the vehicle is an aqueous solution suitable for injection. [The present invention 1062] 1. A method of treating a disease or disorder, comprising: Administering a therapeutically effective amount of any one of the compositions 1046 to 1061 of the present invention to a patient in need thereof; the therapeutic agent is sufficient to treat the disease or disorder, method. [The present invention 1063] The method of claim 1062, wherein the disease or disorder is cancer. [The present invention 1064] The method of claim 1062 or 1063, wherein the therapeutic agent is capable of modulating the immune system to target cancer. [The present invention 1065] The method of any of claims 1062 to 1064, wherein the therapeutic agent generates an immune response against one or more cancer cells. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0035] [Figure 1]Figure 1A-C show the characterization of the ultra-pH-sensitive response of biodegradable UPS copolymers. (Figure 1A) pH changes as a function of the protonation degree of PEO-bP(MAC-SR) copolymers with different tertiary amine substituents. The data of PEO123-bP(MAC-SDMA)135 are presented separately in Figure 4. (Figure 1B) pKa values ​​are inversely correlated with the log P of the repeating units of the P(MAC-SR) segment (neutral / deprotonated state). Blue and pink dots indicate copolymers with linear and cyclic dialkyl tertiary amines as side chains, respectively. (Figure 1C) Sharpness of the pH transition (ΔpH 10%-90%) as a function of log P of PEO-bP(MAC-SR) block copolymers. Commonly used polybases (poly(ethyleneimine), chitosan, polyhistidine, polylysine) are shown for comparison (Li et al., 2016). [Diagram 2] Figures 2A and B show that the phase transition from the soluble unimeric to micellar state drives the ultra-pH-sensitive response of the copolymer. (Figure 2A) Number-weighted hydrodynamic diameter and light scattering count rate as a function of protonation degree during pH titration of PEO123-bP(MAC-SC7A)135 copolymer. (Figure 2B) TEM images and number-weighted hydrodynamic diameter distribution of PEO123-bP(MAC-SC7A)135 at 95% and 85% protonation degree, above and below the critical micellization protonation degree. [Diagram 3]Figures 3A-E show degradation studies of PEO123-bP(MAC-SC7A)135 copolymer in deuterated buffer solutions at pH 6.5 and 7.4. (Figure 3A) Chemical structure of the copolymer and its degradation products. 1H NMR spectra of PEO123-bP(MAC-SC7A)135 copolymer in (Figure 3B) pH 6.5 and (Figure 3C) pH 7.4 deuterated buffer solutions over time. For clarity, only selected regions of the spectrum are presented. The complete spectrum is shown in Figures 6 and 7. (Figure 3D) Integral ratios of peaks (d1+d2) and (d3+d4) relative to the normalized proton signal of the PEO segment in pH 6.5 deuterated buffer solutions. (Figure 3E) Integral ratios of peaks (d3+d4) relative to the normalized proton signal of the PEO segment in pH 6.5 and 7.4 deuterated buffer solutions. [Figure 4] FIG. 1 shows the pH change as a function of the degree of protonation of PEO123-bP(MAC-SDMA) 135 and PEO123-bP(MAC-SC7A) 135. [Diagram 5] (FIG. 5A) Number-weighted hydrodynamic diameter and light scattering count rate as a function of degree of protonation during pH titration of PEO123-bP(MAC-SDMA)135 copolymer. (FIG. 5B) TEM images and number-weighted hydrodynamic size distribution of PEO123-bP(MAC-SDMA)135 at degrees of protonation of 55% and 45%, above and below the CMPD (approximately 50%). Scale bar: 50 nm. [Figure 6] 1 shows the complete 1H NMR spectrum of PEO123-bP(MAC-SC7A)135 copolymer in pH 6.5 deuterated buffer solution over a period of 55 days. [Figure 7] 1 shows the complete 1H NMR spectrum of PEO123-bP(MAC-SC7A)135 copolymer in pH 7.4 deuterated buffer solution over a period of 55 days. [Figure 8]Normalized IL-2 function in vitro assessed by HEK-Blue™ IL-2 reporter cells: IL-2 concentrations: 200, 50, 10, 2, 0.5, 0.2, 0.05, 0.01 ng / mL; PEG-bP(MAC-SDPA) concentrations: 40, 10, 2, 0.4, 0.1, 0.04, 0.01, 0.002 μg / mL. [Figure 9] Figure 1 shows the B16F10 tumor growth curves under different treatments with IL-2 therapy.Free IL-2 or PEG-bP(MAC-SDPA)-IL-2 was injected intratumorally or intravenously on days 1 and 5 (IL-2: 1 μg per injection; PEG-bP(MAC-SDPA): 200 μg per injection). [Figure 10] Body weight change curves within 11 days are shown. Free IL-2 or PEG-bP(MAC-SDPA)@IL-2 were injected intratumorally or intravenously on days 1 and 5 (IL-2: 1 μg per injection; PEG-bP(MAC-SDPA): 200 μg per injection). [Figure 11] Figure 2 shows the loading efficiency of cGAMP in 1.0 mg / mL biodegradable PEG-bP(MAC-SC7A) micellar nanoparticles. [Figure 12] Figure 12A-C show STING binding and activation of dUPS polymers. Figure 12A Isothermal titration calorimetry (ITC) shows that PSC7A copolymer has much higher binding affinity to STING compared to PSDEA. Kd: apparent dissociation constant. Figure 12B Summary of Kb values ​​(binding affinity, reciprocal of Kd) of different dUPS polymers to STING from ITC experiments. Figure 12C Interferon (IFN) induction levels of THP1-ISG cells incubated with dUPS copolymers (0.5 μM, 48 h) correlated with STING binding affinity. Statistical significance was calculated by t-test: ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 13]Figures 13A and B show that PSC7A nanovaccine can inhibit tumor growth and prolong survival in tumor-bearing mice. C57BL / 6 mice (n = 9 per group) inoculated with 2 x 105 Figure 13A TC-1 or Figure 13B B16-F10 melanoma cells were treated with PBS, tumor antigen peptide alone, PSC7A NPs, low-dose PSC7A vaccine (Vax.Low), or high-dose PSC7A vaccine (Vax.High) at the specific time points indicated above. Vaccination resulted in strong tumor growth inhibition and prolonged survival of these mice. For tumor growth studies, data are presented as mean ± sem, and statistical significance was calculated by t-test: ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 14] Figures 14A and B show that PSC7A nanovaccine inhibits tumor growth in two animal tumor models. Nondegradable PC7A nanovaccine was used for comparison. C57BL / 6 mice (n = 9 per group) inoculated with 2 x 105 Figure 14A TC-1 or Figure 14B B16-F10 melanoma cells were treated with PBS, tumor antigen peptide, PSC7A vaccine, or PC7A vaccine at the specified time points. Data are presented as mean ± sem, and statistical significance was calculated by t-test: ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 15]Figures 15A-C show the short-term safety evaluation of degradable PSC7A NPs and non-degradable PC7A NPs. Figure 15A C57BL / 6 mice (n = 4 per group) were subcutaneously injected with PBS, 300 μg PSC7A NPs, or 300 μg PC7A NPs in the right flank. Figure 15B Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine levels were quantitatively measured by Abcam™ Assay Kit 24 hours after injection. Figure 15C Serum concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) protein levels were quantitatively measured by BD™ CBA Mouse Inflammation Kit 24 hours after injection. Statistical significance was calculated by t-test: ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 16] Histological analysis of major organs for short-term safety evaluation of PSC7A NPs. Representative H&E sections of organs from C57BL / 6 mice after subcutaneous injection of PBS, 300 μg PSC7A NPs or 300 μg PC7A NPs into the right flank. 24 h after administration, mice were killed and organs were harvested. Histology of heart, liver, spleen and kidney did not change significantly after treatment with either polymer compared to PBS. Scale bar: 5 mm (top) and 250 μm (bottom) for each organ group. [Figure 17]Figure 17A-D show the long-term safety evaluation of degradable PSC7A NP and non-degradable PC7A NP. Figure 17A C57BL / 6 mice were subcutaneously injected with PBS, 300 μg PSC7A NP, or 300 μg PC7A NP in the right flank. Inflammatory nodules formed at the injection site in the PSC7A NP and PC7A NP groups. The surface area of ​​the nodules was calculated based on the ellipse model. Figure 17B Changes in nodule surface area over time. Figure 17C Left: Photographs of skin tissues taken from the injection site on day 60; from top to bottom, mice treated with PC7A NP, PSC7A NP, and PBS. Right: Enlarged views of skin tissues from the PSC7A and PC7A groups. Figure 17C Left: H&E staining of formalin-fixed and paraffin-embedded skin tissues (injection site) on day 60; from top to bottom, mice treated with PC7A NP, PSC7A NP, and PBS. Scale bar: 2.5 mm. Center: Magnification of a nodule surrounded by granulomatous inflammation from the PC7A group. Scale bar: 250 μm. Inset: Magnification of the area indicated by the square. Arrows at the bottom left, top left and right indicate macrophages, lymphocytes and neutrophils, respectively. Scale bar: 50 μm. Right: Magnification of skin tissue from the PSC7A group. Scale bar: 250 μm. [Figure 18] Histological analysis of skin tissue (injection site) for long-term safety evaluation of PSC7A NPs. Representative H&E sections of skin tissue (injection site) from C57BL / 6 mice after subcutaneous injection of PBS, 300 μg PSC7A NPs or 300 μg PC7A NPs into the right flank. At 1, 15 and 30 days post-administration, mice were killed and skin tissues were harvested. Inset: magnification of area indicated by square. Dark grey, light grey and medium grey arrows indicate macrophages, lymphocytes and neutrophils, respectively. Scale bars: 2.5 mm (top), 1 mm (middle and bottom), 50 μm (inset). [Figure 19] The sharpness of the pH transition (ΔpH 10%-90%) as a function of log P for dUPS polymers is shown. Commonly used polybases (poly(ethyleneimine), chitosan, polyhistidine, polylysine) are shown for comparison. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Description of Exemplary Embodiments In some aspects, the present disclosure provides polymers that can dissemble above a certain transition pH and form pH-responsive nanoparticles that contain degradable backbones. In some embodiments, these polymers have sharp pH transition values ​​and contain one or more degradable groups that accelerate the clearance of the polymer. These polymers can have a wide range of pH transition points, allowing for a wide range of applications, such as the delivery of drug compounds to specific tissues. In some aspects, the present disclosure provides methods of using these polymers in pH-responsive systems such as those described above, such as delivering therapeutic agents to the body, including therapeutic agents such as immune system regulators that can be used to treat cancer.

[0037] A. Chemical definition When used in the context of chemical groups, "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written -COOH or -CO2H); "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means =NH; and "cyano" means -CN "isocyanyl" means -N=C=O; "azido" means -N3; ​​in the monovalent context "phosphate" means -OP(O)(OH)2 or its deprotonated form; in the divalent context "phosphate" means -OP(O)(OH)O- or its deprotonated form; "mercapto" means -SH; and "thio" means =S; "thiocarbonyl" means -C(=S)-; "sulfonyl" means -S(O)2-; and "sulfinyl" means -S(O)-.

[0038] In the context of chemical formulas, the symbol "-" means a single bond; "=" means a double bond; and "≡" means a triple bond. TIFF0007676331000017.tif5128 is an optional bond, which, if present, may be a single or double bond. TIFF0007676331000018.tif5128 is a single or double bond. Therefore, the formula For example, TIFF0007676331000019.tif10128 is TIFF0007676331000020.tif11128. It is also understood that no atom of such a ring forms part of more than one double bond. It is further noted that the covalent bond symbol "-" does not indicate a preferred stereochemistry when connecting one or two stereogenic atoms. Rather, it is intended to include all stereoisomers and mixtures thereof. The symbol TIFF0007676331000021.tif5128 when pulled vertically across the bond TIFF0007676331000022.tif6128, indicating the point of attachment of that group. It is noted that, to aid the reader in unambiguously identifying the point of attachment, points of attachment are typically only so identified for larger groups. TIFF0007676331000023.tif5128 represents a single bond when the group attached to the thick end of the wedge "goes out of the plane of the paper". TIFF0007676331000024.tif5128 represents a single bond when the group attached to the thick end of the wedge is "into the plane of the paper". TIFF0007676331000025.tif5128 refers to a single bond when the geometry around the double bond (e.g., E or Z) is not defined. Thus, both options, as well as combinations, are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented outward from the plane of the paper.

[0039] The variables may be used as "floating groups" on the ring system, for example, in the formula: When depicted as a group "R" in TIFF0007676331000026.tif14128, the variable may replace any hydrogen atom attached to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is created. When depicted as the group "R" in TIFF0007676331000027.tif16128, unless otherwise stated, the variable may replace any hydrogen atom attached to any of the ring atoms of either of the fused rings. Hydrogens that may be replaced include depicted hydrogens (e.g., hydrogens attached to the nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and optional hydrogens whose presence is dependent on the identity of the ring atom (e.g., hydrogens attached to X when the group X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present in either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the parenthesized R represents a numerical variable. Unless otherwise stated, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0040] For chemical groups and compound classes, the number of carbon atoms in the group or class is as follows: "Cn" or "C=n" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number being as small as possible for the group / class in question. For example, the group "alkyl (C≦8) ", "Cycloalkanediyl (C≦8) ", "Heteroaryl (C≦8) " and "Acyl (C≦8) The minimum number of carbon atoms in the group "alkenyl" is 1. (C≦8) ", "alkynyl (C≦8) " and "heterocycloalkyl (C≦8)The minimum number of carbon atoms in the group "cycloalkyl" is two. (C≦8) The minimum number of carbon atoms in the group "aryl" is 3. (C≦8) " and "Arrangeil (C≦8) It is understood that the minimum number of carbon atoms in the group is 6. "Cn-n'" defines both the minimum (n) and maximum number (n') of carbon atoms in the group. Thus, "alkyl (C2~10) " denotes an alkyl group having from 2 to 10 carbon atoms. These carbon number numerals may precede or follow the chemical group or class that they modify, and may or may not be in parentheses, and do not imply any change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "Olefin C5 " are all synonymous. Except as noted below, to determine whether a group or compound conforms to a specified number of carbon atoms, every carbon atom is counted. For example, the group dihexylamino is dialkylamino. (C=12) An example of a group is dialkylamino. (C=6) Similarly, phenylethyl is not an example of an aralkyl group. (C=8) is an example of a group. When any chemical group or class of compounds defined herein is modified with the term "substituted," any carbon atoms in the moiety that replace a hydrogen atom are not counted. Thus, methoxyhexyl has a total of 7 carbon atoms, but is a substituted alkyl group. (C1~6) is an example of: Unless otherwise stated, any chemical group or compound class recited in a claim without a carbon atom limit has a limit of 12 or less carbon atoms.

[0041] The term "saturated", when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. Furthermore, it does not exclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism, if such bonds are present. When the term "saturated" is used to modify a solution of a substance, it means that the substance cannot be dissolved any more in that solution.

[0042] The term "aliphatic" indicates that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, compound or group. In an aliphatic compound / group, the carbon atoms can be linked together in a straight chain, branched chain, or non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) linked by a single carbon-carbon bond, or unsaturated by one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0043] The term "alkyl" refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl" refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term "alkylidene" refers to the divalent group =CRR', where R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include =CH2, =CH(CH2CH3), and =C(CH3)2. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl as this term is defined above.

[0044] The term "alkenyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched chain acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a straight or branched chain acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while alkenediyl groups are aliphatic, once linked at both ends, this does not prevent the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl as this term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene that has only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule.

[0045] When a chemical group is used with the modifier "substituted," one or more hydrogen atoms are replaced, independently at each occurrence, with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CO2CH2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl, limited to the replacement of hydrogen atoms with halo (i.e., -F, -Cl, -Br, or -I), such that there are no other atoms other than carbon, hydrogen, and halogen. The group -CH2Cl is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl, limited to the replacement of hydrogen atoms with fluoro, such that there are no other atoms other than carbon, hydrogen, and fluorine. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0046] The term "thiol reactive group" refers to a functional group that can undergo reaction with a mercapto group (-SH) to form a covalent bond. Such groups are well known in the literature. Two prototypical groups include haloacetamides, such as iodoacetamide, and maleimides. These groups can be used to react with the mercapto group of a cysteine ​​residue to form a covalent bond to the sulfur atom.

[0047] B. Block Copolymer The pH-responsive micelles and nanoparticles disclosed herein include block copolymers. The block copolymers include hydrophilic and hydrophobic polymer segments. The hydrophobic polymer segments are pH-sensitive. For example, the hydrophobic polymer segments can include ionizable amine groups to make them pH-sensitive. The block copolymers form pH-activatable micellar (pHAM) nanoparticles based on the supramolecular self-assembly of these ionizable block copolymers. At higher pH, the block copolymers assemble into micelles, while at lower pH, ionization of the amine groups in the hydrophobic polymer segments leads to dissociation of the micelles. The ionizable groups can function as hydrophilic / hydrophobic blocks that can be tweaked at different pHs, and can directly affect the dynamic self-assembly of the micelles.

[0048] In some embodiments, the polymer contains one or more blocks that are constructed using a degradable polymer base, such as polycarbonate or polyurea. These components can be used to construct hydrophobic polymer segments. In some embodiments, these polymer segments contain one monomer unit to form a homopolymer. In other embodiments, the polymer segment can contain two or more monomer units to form a polymer segment. When the polymer segment contains two or more monomer units, the monomer units can be a single block of one unit, followed by separate blocks for each additional monomer unit, or the different monomer units can be randomly distributed throughout the polymer block.

[0049] The polymers described herein are shown, for example, in the Summary section above, and in the claims below. They may be made using the synthetic methods outlined in the Examples section. These methods may be further modified and optimized using the principles and techniques of organic chemistry applied by those skilled in the art. Such principles and techniques are taught, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated herein by reference. In addition, the synthetic methods may be further modified and optimized for preliminary, pilot or large-scale production, either batch or continuous, using the principles and techniques of process chemistry applied by those skilled in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development - A Guide for Organic Chemists (2012), which is incorporated herein by reference.

[0050] The polymers described herein may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic form. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of the chemical formula are intended. The polymers may appear as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the polymer may have an S or R configuration. In some embodiments, the polymer may contain two or more atoms with a defined stereochemical orientation.

[0051] The chemical formulas used to represent the polymers described herein typically only show one of several different possible tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is shown for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0052] In addition, the atoms constituting the polymers described herein are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include atoms having the same atomic number but different mass numbers. As common examples, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include tritium and deuterium. 13 C and 14 Includes, but is not limited to, C.

[0053] In some embodiments, the polymers described herein are in salt or non-salt form.With respect to salt form, in some embodiments, the specific anion or cation that forms part of any salt form of the polymers described herein is not critical, as long as the salt as a whole is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are shown in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0054] C. Micellar Systems and Compositions The systems and compositions disclosed herein utilize a single micelle or a series of micelles tuned to different pH levels. Moreover, the micelles have a narrow pH transition range. In some embodiments, the micelles have a pH transition range of less than about 1 pH unit. 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 0.25 pH units, less than about 0.2 pH units, or less than about 0.1 pH units. A narrow pH transition range advantageously provides a sharper pH response that can result in a complete turn-on of the fluorophore with subtle changes in pH.

[0055] The size of the micelles is typically on the nanometer scale (i.e., diameter of about 1 nm to 1 μm). In some embodiments, the micelles have a size of about 10 to about 200 nm. In some embodiments, the micelles have a size of about 20 to about 100 nm. In some embodiments, the micelles have a size of about 30 to about 50 nm.

[0056] D. Targeting part Micelles and nanoparticles may further comprise a targeting moiety. The targeting moiety can be used to target nanoparticles or micelles, for example, to specific cell surface receptors, cell surface markers, or organelles (e.g., nucleus, mitochondria, endoplasmic reticulum, chloroplast, apoplast, or peroxisomes). Such targeting moieties are advantageous in the study of receptor recycling, marker recycling, intracellular pH regulation, and endocytic transport.

[0057] The targeting moiety can be, for example, an antibody or antibody fragment (e.g., Fab' fragment), a protein, a peptide (e.g., signal peptide), an aptamer, or a small molecule (e.g., folic acid). Using methods known in the art, the targeting moiety can be conjugated to the block copolymer (e.g., conjugated to a hydrophilic polymer segment). The choice of the targeting moiety depends on the individual target. For example, antibodies, antibody fragments, small molecules, or binding partners may be more suitable for targeting cell surface receptors and cell surface markers, while peptides, especially signal peptides, may also be more suitable for targeting organelles.

[0058] E. Antigen In some aspects, the present disclosure provides one or more antigen components in the composition. An antigen is a substance that promotes an immune response, so that antibodies are generated specifically against the substance. Some substances are more immunogenic, so that the immune system can easily generate an appropriate immune response, while other substances require help to generate a sufficient immune response to generate antibodies against the antigen. Most cancers may require additional activation to enhance the generation of antibodies against the antigen. Some non-limiting examples of antigens include proteins or fragments of cancer-specific surface proteins or surface proteins that are overexpressed by cancer cells.

[0059] Cancer A variety of different peptides, protein fragments, or proteins can be used as antigens in the compositions. Some non-limiting examples are 5T4, 707-AP (707 alanine proline), 9D7, AFP (alpha-fetal protein), AlbZIP HPG1, alpha5beta1-integrin, alpha5beta6-integrin, alpha-methylacyl-coenzyme A racemase, ART-4 (adenocarcinoma antigen 4 recognized by T cells), B7H4, BAGE-1 (B antigen), BCL-2, BING-4, CA15-3 / CA27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL (antigen recognized by CTL on melanoma), CASP-8 (caspase-8), cathepsin B, cathepsin L, CD19, CD20, CD22, CD2 5, CD30, CD33, CD40, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen), CLCA2 (calcium-activated chloride channel-2), CML28, coactosin-like protein, collagen type XXIII, COX-2, CT-9 / BRD6 (bromodomain testis-specific protein), Cten (C-terminal tensin-like protein), cyclin B1, cyclin D1, cyp-B (cyclophilin B), CYPB1 (cytochrome P450 1B1), DAM-10 / MAGE-B1 (differentiation antigen melanoma 10), DAM-6 / MAGE-B2 (differentiation antigen melanoma 6), EGFR / Her1, EMMPRIN (tumor cell-associated extracellular matrix metalloproteinase inducer), EpCam (epithelial cell adhesion molecule), EphA2 (ephrin type A receptor 2), EphA3 (ephrin type A receptor 3), ErbB3, EZH2 (Zeste homolog enhancer 2), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fr a-1 (Fos-related antigen 1), G250 / CAIX (glycoprotein 250), GAGE-1 (G antigen 1), GAGE-2 (G antigen 2), GAGE-3 (G antigen 3), GAGE-4 (G antigen 4), GAGE-5 (G antigen 5), GAGE-6 (G antigen 6), GAGE-7b (G antigen 7b), GAGE-8 (G antigen 8), GDEP (prostate differentially expressed gene), GnT-V (N-acetylglucosaminyltransferase V), gp100 (glycoprotein 100 kDa), GPC3 (glypican 3),HAGE (helicase antigen), HAST-2 (human signet ring tumor-2), hepsin, Her2 / neu / ErbB2 (human epithelial receptor-2 / neuronal), HERV-K-MEL, HNE (human neutrophil elastase), homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IGF-1R, IL-13Ra2 (interleukin-13 receptor α2 chain), IL-2R, IL-5, immature lamina Nin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205, KK-LC-1 (Kitakyushu lung cancer antigen 1), KM-HN-1, LAGE-1 (L antigen), livin, MAGE-A1 (melanoma antigen A1), MAGE-A10 (melanoma antigen A10), MAGE-A12 (melanoma antigen A12), MAGE-A2 (melanoma antigen A2), MAGE-A3 (melanoma antigen A3), MAGE-A4 (melanoma antigen A4), MAGE-A6 (melanoma antigen A6), MAGE-A9 (melanoma antigen A9), MAGE -B1 (melanoma antigen B1), MAGE-B10 (melanoma antigen B10), MAGE-B16 (melanoma antigen B16), MAGE-B17 (melanoma antigen B17), MAGE-B2 (melanoma antigen B2), MAGE-B3 (melanoma antigen B3), MAGE-B4 (melanoma antigen B4), MAGE-B5 (melanoma antigen B5), MAGE-B6 (melanoma antigen B6), MAGE-C1 (melanoma antigen C1), MAGE-C2 (melanoma antigen C2), MAGE-C3 (melanoma antigen C3), MAGE-D1 (melanoma antigen D 1), MAGE-D2 (melanoma antigen D2), MAGE-D4 (melanoma antigen D4), MAGE-E1 (melanoma antigen E1), MAGE-E2 (melanoma antigen E2), MAGE-F1 (melanoma antigen F1), MAGE-H1 (melanoma antigen H1), MAGEL2 (MAGE-like 2), mammaglobin A, MART-1 / Melan-A (melanoma antigen 1 / melanoma antigen A recognized by T cells), MART-2 (melanoma antigen 2 recognized by T cells), matrix protein 22, MC1R (melanocortin 1 receptor),M-CSF (macrophage colony-stimulating factor gene), mesothelin, MG50 / PXDN, MMP11 (M-phase phosphoprotein 11), MN / CA IX antigen, MRP-3 (multidrug resistance-associated protein 3), MUC1 (mucin 1), MUC2 (mucin 2), NA88-A (NA cDNA clone of patient M88), N-acetylglucosaminyltransferase-V, Neo-PAP (Neo-poly(A) polymerase), NGEP, NMP22, NPM / ALK (nucleophosmin / anaplastic lymphoma kinase fusion protein), NSE (neuron-specific enolase), NY-ESO-1 (New York esophageous 1), NY-ESO-B, OA1 (ocular albinism type 1 protein), OFA-iLRP (carcinoembryonic antigen-immature laminin receptor), OGT (O- N-acetylglucosamine-linked N-acetylglucosamine transferase gene), OS-9, osteocalcin, osteopontin, p15 (protein 15), p15, p190 minor bcr-abl, p53, PAGE-4 (prostate GAGE-like protein 4), PAI-1 (plasminogen activator inhibitor 1), PAI-2 (plasminogen activator inhibitor 2), PAP (prostatic acid phosphatase), PART-1, PATE, PDEF, Pim-1-kinase, Pin1 (prostate glucosamine transferase gene), pyrimidine isomerase), POTE, PRAME (preferentially expressed antigen in melanoma), prostein, proteinase 3, PSA (prostate-specific antigen), PSCA, PSGR, PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal antigen), RHAMM / CD168 (hyaluronan-mediated motility receptor), RU1 (renal ubiquitous 1), RU2 (renal ubiquitous 1), S-100, SAGE (sarcoma antigen), SART-1 (squamous epithelial cell rejection antigen 1), SART-2 (squamous epithelial cell rejection antigen 2), tumor 1), SART-3 (squamous cell carcinoma antigen rejection tumor 1), SCC (squamous cell carcinoma antigen), Sp17 (sperm protein 17), SSX-1 (synovial sarcoma X breakpoint 1), SSX-2 / HOM-MEL-40 (synovial sarcoma X breakpoint), SSX-4 (synovial sarcoma X breakpoint 4), STAMP-1, STEAP (six transmembrane epithelial antigen prostate), surviving, survivin 2B (intron 2 retaining survivin), TA-90, TAG-72, TARP,TGFb (TGFβ), TGFbRII (TGFβ receptor II), TGM-4 (prostate-specific transglutaminase), TRAG-3 (taxol resistance-associated protein 3), TRG (testin-related gene), TRP-1 (tyrosine-related protein 1), TRP-2 / 6b (TRP-2 / novel exon 6b), TRP-2 / INT2 (TRP-2 / intron 2), Trp-p8, tyrosinase, UPA (urokinase-type plasminogen activator), VEGF (vascular endothelial growth factor), VEGFR-2 / FLK-1 (vascular endothelial proliferation factor) factor receptor 2), WT1 (Wilms tumor gene), or, for example, α-actinin-4 / m, ARTC1 / m, bcr / abl (breakpoint cluster region-Abelson fusion protein), β-catenin / m (β-catenin), BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m (cell division cycle 27), CDK4 / m (cyclin-dependent kinase 4), CDKN2A / m, CML66, COA-1 / m, DEK-CAN (fusion protein), EFTUD2 / m, ELF2 / m (elongation factor 2), ETV6-AML1 (Ets heterologous gene 6 / acute myeloid leukemia 1 gene fusion protein), FN1 / m (fibronectin 1), GPNMB / m, HLA-A*0201-R170I (arginine to isoleucine exchange at residue 170 of the α-helix of the α2 domain in the HLA-A2 gene), HLA-A11 / m, HLA-A2 / m, HSP70-2M (heat shock protein 70-2 mutant), KIAA0205 / m, K-Ras / m, LDLR-FUT (LDR-fucosyltransferase fusion protein), MART2 / m, M E1 / m, MUM-1 / m (melanoma ubiquitous mutant type 1), MUM-2 / m (melanoma ubiquitous mutant type 2), MUM-3 / m (melanoma ubiquitous mutant type 3), myosin class I / m, neo-PAP / m, NFYC / m, N-Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARα (promyelocytic leukemia / retinoic acid receptor α), PRDX5 / m, PTPRK / m (receptor protein tyrosine phosphatase kappa), RBAF600 / m, SIRT2 / m, SYT-SSX-1 (synaptotagmin I / synovial sarcoma X fusion protein),The antigen may include a mutant antigen expressed in a cancer disease selected from the group including, but not limited to, SYT-SSX-2 (synaptotagmin I / synovial sarcoma X fusion protein), TEL-AML1 (translocation Ets family leukemia / acute myeloid leukemia 1 fusion protein), TGFβRII (TGFβ receptor II), and TPI / m (triose phosphatase isomerase).

[0060] F. Kit The present disclosure also provides a kit. Any of the components disclosed herein can be incorporated into the kit. In some embodiments, the kit comprises the pH-responsive system or composition described above.

[0061] The kit generally includes at least one vial, test tube, flask, bottle, syringe or other container into which the components can be placed, and preferably appropriately dispensed. If there are more than one component in the kit, the kit also generally contains a second, third or other additional container into which the additional components can be placed separately. However, various combinations of components may be included in one container. In some embodiments, all of the micelle populations in the series are combined in a single container. In other embodiments, some or all of the micelle populations in the series are provided in separate containers.

[0062] The kits of the present disclosure also typically include a package containing various containers in tightly sealed containers for commercial sale. Such packages may include cardboard or injection or blow molded plastic packages in which the desired containers are held. The kits may also include instructions for using the kit components. The instructions may include variations that can be implemented. EXAMPLES

[0063] G. Working Example The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in implementing the present disclosure, and therefore may be considered to constitute preferred modes for its implementation. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0064] Example 1: Synthesis Characterization, Materials, and Methods 1.Material All reagents were purchased from commercial sources or synthesized and used without further purification unless otherwise specified. They were poly(ethylene glycol) methyl ether (mPEG 5k -OH,M n = 5.4 × 10 3 g / mol, 1The cations of the benzene rings were determined by H NMR, 1-(3,5-bis-trifluoromethyl-phenyl)-3-cyclohexylthiourea (TU, synthesized) (Natarajan et al., 2005), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, ≥99%, Sigma-Aldrich), dipropylamine (DPA, 99%, Sigma-Aldrich), dibutylamine (DBA, ≥99.5%, Sigma-Aldrich), pyrrolidine (C5A, ≥99%, Sigma-Aldrich), piperidine (C6A, ≥99.5%), hexamethyleneimine (C7A, 99%, Sigma-Aldrich), ethylene sulfide (98%, Sigma-Aldrich), and 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%, Sigma-Aldrich). 2-Dimethylaminoethanethiol hydrochloride (DMA-SH·HCl, 95%) and 2-diethylaminoethanethiol hydrochloride (DEA-SH·HCl, 95%) were purchased from Sigma-Aldrich. Other aminothiol hydrochloride molecules (shown below) were synthesized as reported ( Hao et al., 2015 ). TIFF0007676331000028.tif56128

[0065] Synthesis of PEO-b-PMAC copolymer First, 5-methyl-5-allyloxycarbonyl-1,3-dioxane-2-one (MAC) monomer was synthesized as reported (Hu et al., 2007). PEO-b-PMAC copolymer was prepared by polymerization of mPEG as an initiator. 5k The compound was synthesized by ring-opening polymerization (ROP) using -OH ([monomer] / [initiator] = 200). Typically, in a glove box filled with purified argon, 0.4 g mPEG was added to a Schlenk reaction flask. 5k-OH, 3.2 g MAC monomer and 16.0 mL dichloromethane (DCM). After three freeze-pump-thaw cycles, 0.6 g TU and 0.16 mL DBU were introduced to initiate the polymerization. The reaction was placed in an oil bath at 30°C for 15 h and then quenched by the addition of benzoic acid. The DCM solvent was removed by evaporation and the concentrated residue was precipitated into an excess of cold ether. The purification process was repeated twice to remove unreacted starting materials and impurities. The resulting PEO-b-PMAC copolymer was analyzed by NMR spectroscopy at 400 MHz. 1 Characterized by H NMR, gel permeation chromatography (GPC, Viscotech GPCmax, PLgel 5 μm MIXED-D column, Polymer Labs, 1% v / v TEA in THF was used as eluent at 1.0 mL / min). PEG 123 -b-PMAC 125 About TIFF0007676331000029.tif32150

[0066] Synthesis of PEO-bP(MAC-SR·HCl) copolymer PEO-bP(MAC-SR·HCl) copolymer was synthesized by the thiol-ene reaction of allyl-containing PEO-b-PMAC with aminothiol hydrochloride. In the following, we chose the synthesis of PEO-bP(MAC-SDEA·HCl) as an example. First, 0.1 g PEO 123 -b-PMAC 125 (0.419 mmol) was dissolved in 15 mL DMF in a quartz flask and stirred under nitrogen for 10-20 min. Then, 1.06 g DEA-SH·HCl (6.29 mmol) and 21.5 mg DMPA (0.084 mmol) were added to the flask. After purging with nitrogen for another 20 min, the flask was placed under UV light (365 nm) to initiate the reaction. After 12 h, the reaction mixture was dialyzed in distilled water and lyophilized to obtain a white powder. A series of PEO-bP(MAC-SR·HCl) copolymers were synthesized. 1 The results were confirmed by H NMR and GPC, and are summarized in Table 1.

[0067] Table 1. Characterization of PEO-bP(MAC-SR HCl) copolymer TIFF0007676331000030.tif102145 a The number of repeat units for the PEO-b-PMAC copolymer precursor is 140. b The number of repeat units for the PEO-b-PMAC copolymer precursor is 125. c M w,GPC , M n,GPC and PDI (M w,GPC / M n,GPC ) was obtained by using polystyrene as standards and THF (1% v / v TEA) as the eluting solvent in GPC.

[0068] TIFF0007676331000031.tif161150A small percentage of the allyl groups of PMAC did not react completely with DBA-SH·HCl due to steric hindrance of the dibutyl groups. TIFF0007676331000032.tif109150

[0069] Preparation of micellar nanoparticles PEO-bP (MAC-SC7A) is used as an example. In a typical procedure, 10 mg PEO 123 -bP(MAC-SC7A·HCl) 135 The copolymer was dissolved in distilled water containing 150 mM NaCl. NaOH solution was added to adjust the final pH value to above 8.0. Excess NaOH and salts were removed by three cycles of ultracentrifugation using a 3000 Da molecular weight cutoff centrifuge tube. Distilled water was added to the micelle solution to adjust the polymer concentration to 1.0 mg / mL.

[0070] pH titration experiment PEO-bP (MAC-SC7A) is used as an example. In a typical experiment, 10 mg PEO was added to achieve a polymer concentration of 1.0 mg / mL. 123 -bP(MAC-SC7A·HCl) 135 The copolymer was first dissolved in 10 mL distilled water. NaCl was added to adjust the salt concentration to 150 mM. PEO was then added. 123 -bP(MAC-SC7A·HCl) 135 To fully deprotonate the copolymer, NaOH solution was added. pH titration was performed by adding small amounts (1 μL each) of 0.5 M HCl solution under stirring. pH values ​​were measured using a Mettler Toledo pH meter equipped with a microelectrode. The pH decrease over the entire range was monitored as a function of the total added HCl volume. The fully protonated (100% degree of protonation) and deprotonated (0% degree of protonation) states were determined by the two extreme points of the first derivative of the pH titration curve. At the selected degree of protonation, 100 μL of the polymer solution was removed for dynamic light scattering measurements (DLS, Malvern Nano-ZS model, He-Ne laser, λ = 633 nm). Similar titration procedures were followed for other PEO-bP(MAC-SR) copolymers.

[0071] TEM images of different copolymers To achieve a polymer concentration of 1.0 mg / mL, PEO 123 -bP(MAC-SC7A·HCl) 135 was first dissolved in distilled water. NaCl was added to adjust the salt concentration to 150 mM. Based on the titration coordinate, a corresponding amount of 0.5 M NaOH was added to adjust the degree of protonation to 95% and 85%. The polymer solution was diluted to 0.2 mg / mL and dropped onto a copper grid. The grid was dried and the grid was rinsed for a few seconds using distilled water to remove NaCl, after which phosphotungstic acid (PTA) was added for negative staining. Similarly, PEO with a degree of protonation of 55% and 45% was diluted to 0.2 mg / mL and dropped onto a copper grid. 123 -bP(MAC-SDMA) 135 The copolymers were imaged by TEM.

[0072] PEO in pH 6.5 and 7.4 buffers 123 -bP(MAC-SC7A) 135 Decomposition study of Deuterated phosphate buffer solutions of pH 6.5 and 7.4 were prepared with Na2HPO4 and NaH2PO4 (50 mM) in D2O. NaCl was added to reach a final concentration of 150 mM. For the pH 6.5 solution, 5.0 mg PEO was added to achieve a polymer concentration of 5.0 mg / mL. 123 -bP(MAC-SC7A·HCl) 135 The copolymer was dissolved in 1.0 mL deuterated phosphate buffer solution. The pH of the polymer solution was further adjusted to 6.5 with concentrated NaOD and DCl solutions. The tube was then sealed and placed in a 37° C. shaker at a speed of 150 rpm. At a certain time, the polymer solution was 1 The polymer was transferred into an NMR tube for H NMR measurement. The pH of the polymer solution was adjusted every other day. The pH 7.4 solution study was carried out following a similar procedure.

[0073] Isothermal Titration Calorimetry (ITC) ITC was used to measure the binding affinity between STING dimers and dUPS copolymers using a Marvin ITC200 microcalorimeter. Titrations were performed at 20°C in a buffer containing 25 mM HEPES (pH 6.5). Titration traces were integrated by NITPIC and curves were fitted by SEDFIT. Figures were generated using GUSSI (biophysics.swmed.edu / MBR / software.html).

[0074] STING reporter experiments THP1-ISG cells (5 × 10 5Cells (1000 x 1000 cells / mL) were incubated with phorbol 12-myristate 13-acetate (PMA) in complete medium (RPMI-1640, 10% fetal bovine serum, 100 U / mL penicillin G sodium and 100 μg / mL streptomycin) for 48 hours at 37°C in 5% CO2 and normal O2 levels, and were supplemented with fresh medium for an additional 24 hours. Cells were then incubated with fresh medium containing different dUPS copolymers (0.5 μM) for 48 hours. The levels of IRF-inducible Lucia luciferase in cell culture supernatants were assessed using the luciferase detection reagent QUANTI-Luc™.

[0075] PSC7A vaccine and tumor therapy experiments Nanovaccines were made by physically mixing tumor-specific antigen peptides and PSC7A nanoparticles. A non-degradable PC7A-based nanovaccine was used for comparison. 2 × 10 5 TC-1 cells or B16F10 melanoma cells were inoculated subcutaneously. In the TC-1 tumor model, mice were subcutaneously injected into the base of the tail with PBS, E7p alone (0.5 μg), PSC7A NP alone (30 μg), low-dose PSC7A nanovaccine (0.1 μg E7p in 6 μg PSC7A NP), high-dose PSC7A nanovaccine (0.5 μg E7p in 30 μg PSC7A NP), and high-dose PC7A nanovaccine (0.5 μg E7p in 30 μg PC7A NP) on days 8, 14, and 24 after inoculation. In the B16F10 tumor model, mice were subcutaneously injected into the base of the tail with PBS, Trp1,2 alone (0.5 μg Trp1,2, or ... 214-237 and 0.5 μg Trp2 173-196), PSC7A NP alone (30 μg), low dose PSC7A nanovaccine (0.1 μg Trp1 and 0.1 μg Trp2 in 6 μg PSC7A NP), high dose PSC7A nanovaccine (0.5 μg Trp1 and 0.5 μg Trp2 in 30 μg PSC7A NP), and high dose PC7A nanovaccine (0.5 μg Trp1 and 0.5 μg Trp2 in 30 μg PC7A NP) were injected subcutaneously into the base of the tail of mice. Tumor growth was then measured using digital calipers and expressed as 0.5 × length × width. 2 The tumor volume was calculated as follows: 3 Mice were sacrificed when

[0076] statistical analysis Statistical analysis was performed using Origin and Graphpad Prism. Data are expressed as mean ± sem. Data were analyzed by t-test and P < 0.05 ( *** P < 0.001, ** P < 0.01, * P < 0.05) were considered statistically significant.

[0077] Example 2: Synthesis and characterization of a library of pH-responsive biodegradable polymers Scheme 1 shows the synthesis of biodegradable UPS copolymers using ring-opening polymerization (ROP) (Chen et al., 1997; Brannigan and Dove, 2017; Feng et al., 2012). First, the allyl-functionalized block copolymer, poly(ethylene oxide)-b-poly(5-methyl-5-allyloxycarbonyl-1,3-dioxane-2-one) (PEO-b-PMAC), was reacted with methoxy-terminated polyethylene glycol (PEO) (mPEG) as a macroinitiator in dichloromethane (DCM). 5k -OH,M n = 5.4 × 10 3 g / mol, 1The product was synthesized using 1,3-dioxane-2-one (MAC) as the cyclic monomer and 1-(3,5-bis-trifluoromethyl-phenyl)-3-cyclohexylthiourea (TU) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as organic cocatalysts. The reaction mixture was heated at 30° C. for 15 h. 1 PEO-b-PMAC copolymers with 125-140 repeat units in the PMAC segment were obtained as determined by H NMR. To impart pH sensitivity, PEO-b-PMAC was further reacted with a series of protonated tertiary amines (R·HCl) via thiol-ene reaction under UV light (365 nm, Scheme 1). As a result, a library of biodegradable ultra-pH-sensitive copolymers PEO-bP(MAC-SR·HCl) was synthesized in the protonated state (Table 1). PSR was used below to refer to the block copolymer PEO-bP(MAC-SR·HCl) and its deprotonated state.

[0078] Scheme 1. Synthesis of PEO-bP(MAC-SR·HCl) via ring-opening polymerization and thiol-ene reaction. The final copolymer consists of hydrophilic PEO segments, a biodegradable polycarbonate backbone, and an ionizable tertiary amine that confers pH sensitivity. TIFF0007676331000033.tif54150

[0079] pH titrations of a series of newly synthesized dUPS copolymers (1.0 mg / mL) were performed in the presence of 150 mM NaCl to mimic physiological salt concentrations. Data are presented as pH versus the degree of protonation of the tertiary amine residues on the copolymers. The degree of protonation was calculated as the molar percentage of total amines in the protonated form. The apparent pK of each copolymer was a was measured as the pH at which the degree of protonation was 50%. To evaluate the sharpness of the pH transition, ΔpH 10%-90% was measured for each copolymer.

[0080] The result is PEO 123 -bP(MAC-SDMA) 135 It is shown that, except for PEO (copolymer with dimethylamine side chains) (PSDMA), all other copolymers exhibited ultra-pH-sensitive behavior in most of the pH titration coordinate (Figure 1A). The ultra-sensitive pH response is represented by a prominent pH plateau over a wide range of protonation degrees, especially from 10% to 90%, demonstrating a strong pH buffering effect within a narrow pH range. Interestingly, PEO 123 -bP(MAC-SDMA) 135 exhibited a bi-segmented pH response, with a broad response above 50% protonation and a narrow response below 50% (see FIG. 4). 10%-90% The values ​​were below 0.5 for these copolymers. A higher ΔpH was observed for PSDMA with dimethylamine side chains. 10%-90% Commonly used polybases (e.g., poly(ethyleneimine), chitosan, polyhistidine, polylysine) have a ΔpH 10%-90% The polymers exhibited a broad pH response, with pH >2 (Figure 19) (Li et al., 2016). Furthermore, non-degradable PMMA copolymers with dimethylamine side chains exhibited a broad pH response across the entire pH titration coordinate (Li et al., 2016).

[0081] Apparent pK of PEO-bP(MAC-SR HCl) copolymer a The pK values ​​showed an inverse correlation with the hydrophobicity of the tertiary amine substituents (Figure 1B). The octanol-water partition coefficients (log P) of the repeating units of the P(MAC-SR) segment (neutral / deprotonated state) were used to quantify molecular hydrophobicity. The data were presented as apparent pK values ​​as a function of log P for copolymers containing cyclic or linear amines. a The more hydrophobic side chains have a lower pK a The pK values ​​of these copolymers were a Values ​​covered a wide range of physiological pH from 7.7 to 5.2.

[0082] ΔpH as a function of log P 10%-90% The plot of indicates the existence of a hydrophobicity threshold for the ultra-pH sensitive response (FIG. 1C). For copolymers with log P > 2, ΔpH 10%-90% The values ​​were less than 0.5. PEO with log P < 2 123 -bP(MAC-SDMA) 135 For a higher ΔpH 10%-90% values ​​(1.2) were observed. For comparison, the most commonly used polybases (e.g., poly(ethyleneimine), chitosan, polyhistidine, polylysine) are hydrophilic with log P < 2 and exhibit a wide pH response. Poly(ethyleneimine) exhibited the highest ΔpH due to its strong hydrophilicity (log P = -3.8) and negative cooperativity in the protonation process. 10%-90% value (5.6). These results are consistent with non-degradable PMMA polymers and suggest that molecular hydrophobicity is a common driver for the extreme pH sensitivity in both systems (Li et al., 2016).

[0083] Two copolymers PEO 123 -bP(MAC-SC7A) 135 (PSC7A) and PEO 123 -bP(MAC-SDMA) 135 For (PSDMA), its effect on hydrophobicity-driven phase transition (i.e., micellization) and pH sensitivity was investigated using log P values ​​above and below the hydrophobicity threshold. 123 -bP(MAC-SC7A) 135 For PEO, dynamic light scattering (DLS) results during pH titration (Figure 2A) indicate that when the degree of protonation was above 90%, the polymer chains existed as unimers with hydrodynamic diameters less than 10 nm. When the degree of protonation was reduced below 90%, micelles began to form, as indicated by an increase in the scattering count rate. The critical micellization protonation degree (CMPD) was defined as the degree of protonation below which the polymer chains began to self-assemble. 123 -bP(MAC-SC7A) 135For PEO with a protonation degree of 95% and 85%, the CMPD value is 90%. 123 -bP(MAC-SC7A) 135 Transmission electron microscopy (TEM) images and number-weighted hydrodynamic size distributions (Figure 2B) further supported the phase transition across the CMPD: the micelle diameter remained around 45 nm below a protonation degree of 90%.

[0084] Micellization-induced hyper-pH sensitivity of PEO 123 -bP(MAC-SDMA) 135 This polymer exhibits a bi-segmented pH response, with a broad response above 50% protonation and a narrow response below 50% (Figure 5). Above 50% protonation, PEO 123 -bP(MAC-SDMA) 135 remained as unimers and showed a wide pH response. A dramatically sharp pH response was observed below 50% protonation. The ultra-pH-sensitive response coincided with the formation of micelles across the CMPD, allowing direct evidence of the phase-transition-induced ultra-pH-sensitive response. Interestingly, the non-degradable PMMA polymer with dimethylamine side chains did not show any phase transition behavior during the entire course of pH titration. These results suggest that the polycarbonate backbone PMAC is more hydrophobic than PMMA and also contributes to micelle formation.

[0085] PEO 123 -bP(MAC-SC7A) 135 was selected as a representative biodegradable copolymer to investigate its degradation properties at pH 7.4 and 6.5, which mimic the normal physiological pH environment and the early endosomal pH environment, respectively. 123 -bP(MAC-SC7A) 135 Apparent pK aThe pH of the copolymer is 6.9, so that the copolymer exists in a micellar or protonated unimer state at pH 7.4 and 6.5, respectively. The copolymer was prepared at 5.0 mg / mL in deuterated phosphate buffer solution (50 mM) containing NaCl (150 mM). NaOD or DCl solution was added to adjust the pH to 6.5 or 7.4. Figure 3A shows the PEO copolymer. 123 -bP(MAC-SC7A) 135 The structures of the copolymer and its degradation products are shown. During the degradation process, the copolymer may degrade into oligomers, monomers, PEO segments, and carbon dioxide, all due to hydrolytic cleavage of the polycarbonate ester backbone (left structural circle in FIG. 3A). Further hydrolysis of the ester groups on the side chains (right structural circle in FIG. 3A) may lead to additional degradation products, 2,2-bis(hydroxymethyl)propionic acid (bis-HPA) and 3-(2-azepan-1-yl-ethylsulfanyl)-propanol. To monitor the formation of degradation products over 55 days in both pH solutions, 1 H NMR was used. The proton signal of the PEO segment did not change over time, and its peak integral was used as an internal reference to other proton peaks for quantification.

[0086] Figure 3B shows the effect of PEO at pH 6.5 over time. 123 -bP(MAC-SC7A) 135Figure 3A shows the degradation profile of copolymer 100%. Since the copolymer existed as protonated unimers in solution, all proton peaks were visible at time zero (e.g., proton signals corresponding to c1, d1, and e1). Within the first few days, new peaks (c3, c4, d2, and d3) formed and their intensity increased over time. At day 25, most of the copolymer polymer appeared to have degraded to monomeric structures (yellow panel in Figure 3A) and PEO. Additional hydrolysis from days 25 to 55 shows a small proportion of the further degradation product bis-HPA (d4). Quantification of the degradation rate was performed by analyzing the decrease in peak intensity of d1 and d2 (from polymeric and oligomeric states, respectively) or the increase in d3 and d4 (monomer and bis-HPA). The data show that two sets of curves cross simultaneously at 50% relative peak intensity on day 12 (Figure 3D). As used herein, t 1 / 2 is defined as the half-life at which 50% of the copolymer is converted to monomer.

[0087] The degradation profile at pH 7.4 is more complex due to the formation of micelles. At time zero, only the PEO peak is observed. 1 H NMR was visible (Figure 3C), because the micelle formation of the P(MAC-SC7A) segments caused signal suppression due to fast transverse relaxation of the proton signals. Over time, proton signals from degraded monomer and bis-HPA were observed, albeit at a slower rate of formation. Interestingly, at day 55, a higher ratio of bis-HPA to MAC monomer was seen. Quantitative analysis of the monomer and bis-HPA peaks (d3+d4) revealed that the t 1 / 2 (Figure 3E). The slower degradation rate at pH 7.4 compared to pH 6.5 may be due to several factors, including limited water penetration and access to PMAC due to the hydrophobic micellar core or reduced acid-catalyzed hydrolysis of carbonate bonds at pH 7.4.

[0088] The polycarbonate backbone is hydrolytically active, allowing the polymer to spontaneously degrade in an aqueous environment into biocompatible PEO segments and small molecules. The degradation rates demonstrate that hydrolysis occurs at a slower rate at pH 7.4 than at 6.5 (t 1 / 2 are 27 and 12 d, respectively).

[0089] Example 3: Encapsulation of IL-2-loaded micelles PEG-bP(MAC-SDPA) (pKa=6.1) micelles were developed to deliver IL-2, a T cell growth factor, to the tumor microenvironment. In a typical procedure, 0.2 mg PEG-bP(MAC-SDPA) was dissolved in 0.05 mL methanol and then dropped into 0.5 mL PBS (pH 7.4) to form empty micelles. Methanol was removed by ultrafiltration (100 kDa, 5000 rpm / 15 min, for two times). The micelles were resuspended in PBS and then mixed with different amounts of human recombinant IL-2 protein.

[0090] IL-2 loading efficiency was evaluated by mixing 20 μg (10%), 10 μg (5%), or 1 μg (0.5%) IL-2 with PEG-bP(MAC-SDPA) micelles. Free IL-2 was removed from IL-2 loaded PEG-bP(MAC-SDPA) micelles by ultrafiltration (100 kDa, 5000 rpm / 15 min, for two runs). The filtrate was collected for IL-2 concentration determination by HPLC. In all cases, more than 90% of IL-2 protein was found to be loaded into PEG-bP(MAC-SDPA) micelles. The IL-2 content in PEG-bP(MAC-SDPA) micelles was calculated to be 8.26%, 4.31%, and 0.45%, respectively. The particle size of IL-2-PEG-bP(MAC-SDPA) was about 55 nm as determined by dynamic light scattering analysis.

[0091] A. Assessment of IL-2 Effects In Vitro IL-2 function was examined using HEK-Blue™ IL-2 reporter cells, which are designed to monitor the activation of the JAK-STAT pathway induced by IL-2. Briefly, cells were rinsed with pre-warmed PBS, detached from flasks, and cell suspensions were prepared at approximately 280,000 cells / mL. Then, 20 μL of free IL-2 or IL-2-loaded PEG-bP (MAC-SDPA) were added to flat-bottom 96-well plates. Samples were incubated with 180 μL of cell suspension per well at 37° C. in a CO2 incubator (IL-2 concentrations: 200, 50, 10, 2, 0.5, 0.2, 0.05, 0.01 ng / mL; PEG-bP (MAC-SDPA) concentrations: 40, 10, 2, 0.4, 0.1, 0.04, 0.01, 0.002 μg / mL). After 24 hours, 20 μL of induced HEK-Blue™ IL-2 cell supernatant per well was added into another 96-well plate and mixed with 100 μL QUANTI-Blue™ detection solution. The plate was incubated in a 37° C. incubator for 1 hour, and then SEAP levels were determined using a spectrophotometer at 630 nm.

[0092] The data show that the biological effect of PEG-bP(MAC-SDPA)-encapsulated IL-2 was dramatically enhanced compared to free IL-2 (Figure 8). EC 50 were 0.8 ng / mL and 15 ng / mL, respectively. Micelle-delivered IL-2 showed approximately 20-fold higher potency compared to free IL-2.

[0093] B. Evaluation of Antitumor Efficacy in Vivo The tumor growth inhibitory effects of free IL-2 and PEG-bP(MAC-SDPA)-IL-2 in the B16F10 melanoma tumor model were evaluated. C57bl / 6j mice were first inoculated with B16F10 cells (2.5 × 10 in 100 μL PBS). 5 The tumors were 50–80 mm 3When the mice had grown to a size of 100 μg / injection, they were randomly divided into 5 groups. Free IL-2 or PEG-bP(MAC-SDPA)-IL-2 was injected intratumorally or intravenously on days 1 and 5 (IL-2: 1 μg per injection; PEG-bP(MAC-SDPA): 200 μg per injection).

[0094] As shown in Figure 9, PEG-bP(MAC-SDPA)-IL-2 after iv injection showed improved tumor growth inhibition compared to iv injection of free IL-2 at the same dose. The antitumor effect of IL-2 after it injection was also enhanced after loading in PEG-bP(MAC-SDPA) micelles compared to free IL-2. All treatment groups showed tumor growth inhibition compared to the PBS control.

[0095] The body weight of mice in the PBS and free IL-2 it groups increased slightly after 1 week, likely due to tumor growth. For the PEG-bP(MAC-SDPA)-IL-2 groups (both iv and it), no obvious weight loss was observed during treatment. However, after iv injection, free IL-2 caused some temporary loss in mice (Figure 10), indicating a potential side effect of systemic administration of free IL-2.

[0096] Example 4: Encapsulation of cGAMP micelles cGAMP is an endogenous second messenger and high affinity ligand that triggers type I IFN production through the STING pathway. It is an anionic, highly water-soluble molecule, and its activity and therapeutic effect are limited by low bioavailability and poor drug-like properties. For efficient cytoplasmic delivery of cGAMP, we developed nanoparticles based on our biodegradable PEG-bP(MAC-SC7A) micelles.

[0097] In a typical formulation process, PEG-bP (MAC-SC7A·HCl) was first dissolved in 5% glucose aqueous solution to achieve a polymer concentration of 1.0 mg / mL. A small amount of HCl was then added, followed by different amounts of cGAMP (2%, 5% and 10% of polymer, w / w). A specific volume of NaOH was added to the solution to adjust the final pH to about 7.4. After ultrafiltration (10 kDa), the filtrate was collected and the amount of unloaded cGAMP was measured by HPLC. The corresponding cGAMP loading efficiency was calculated as follows: Loading efficiency (%) = (weight of cGAMP loaded / weight of total cGAMP) × 100%.

[0098] The results are shown in Figure 11. The loading efficiency of cGAMP in PEG-bP(MAC-SC7A) nanoparticles can reach as high as 90% (2% cGAMP at 1.0 mg / mL PSC7A). The loading efficiency of the other two formulations also reaches over 80%, which is very high for cGAMP-loaded nanoparticle formulations. Meanwhile, the formulations are very stable. The loading efficiency does not change much even after 24 hours.

[0099] Example 5: STING Activation, Antigen Delivery, and T Cell Therapy of Cancer Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated homodimeric protein that plays an important role in innate immunity (Barber, 2015; Ishikawa and Barber, 2008). STING activation mediates the CD8+ / CD9+ response to cancer. +This results in upregulation of type I interferon (IFN) enhancing T cell responses (Baccala et al., 2007; Fuertes et al., 2013; Zitvogel et al., 2015). Previously, a non-degradable polymeric nanoparticle, PC7A NP, was reported that allows efficient encapsulation and cytoplasmic delivery of tumor antigens to lymph node-resident dendritic cells. This polymer also binds and activates STING, turning on the co-stimulatory pathway (CD80 / CD86) for the generation of antigen-specific T cells (Luo et al., 2017).

[0100] In this study, the binding affinity of a series of dUPS copolymers (PSC7A, PSC6A, PSC5A, and PSDEA) was first evaluated against the C-terminal domain of STING (139-397 AA). pH 6.5, where all copolymers remain as cationic unimers in solution, was selected for binding studies. Isothermal calorimetry (ITC) results showed that the PSC7A copolymer exhibited a dissociation constant (K d ) had the highest binding affinity to STING (Figure 12A). The other two copolymers with cyclic tertiary amines, PSC6A and PSC5A, had K d The copolymer PSEDA, which has a linear tertiary amine, had negligible binding to STING. The K of non-degradable PC7A to STING dThe value was 72 nM, higher than that by PSC7A. THP1-ISG cells were used to evaluate STING activation after treatment with different copolymers (0.5 μM) for 48 h (Figure 12C). THP1-ISG cells were transfected with a luciferase reporter gene under the control of an interferon regulatory factor inducible promoter. Upon STING activation, secretion of type I IFN activates luciferase expression for luminescence detection. The results show that IFN induction was elevated with dUPS copolymers bearing cyclic tertiary amines compared to the linear analogues. Notably, PSC7A copolymers increased IFN induction up to 14-fold, which correlated with the highest binding affinity to STING by ITC measurements.

[0101] Based on the STING binding and activation assays, the PSC7A copolymer was selected for subsequent T cell vaccine studies (Figure 13). PSC7A nanoparticles were first fabricated and then mixed with tumor-specific antigen peptides. Human papillomavirus (HPV) E6 / E7 transfected TC-1 and mouse B16-F10 melanoma tumor models were used. Tumor cells (2 × 10 5 In the TC-1 model, E7 peptide antigen was administered to mice. TIFF0007676331000034.tif4128 was used. Different groups were subcutaneously injected at the base of the tail on days 8, 16 and 24 after tumor inoculation (shown in FIG. 13A). PBS, E7p and PSC7A NP alone groups were used as controls. The results show that the E7p and PSC7A NP alone groups had a minimal tumor growth inhibition response compared to the PBS control. Most animals were lost within 30 days after tumor inoculation. In contrast, the E7p-PSC7A NP group resulted in dramatically improved tumor growth inhibition and extended survival time. The low-dose PSC7A vaccine group (0.1 μg E7p in 6 μg PSC7A NP) resulted in >50% animal survival 50 days after tumor inoculation, while the high-dose vaccine group (0.5 μg E7p in 30 μg PSC7A NP) had a complete survival outcome (FIG. 13A). For the B16-F10 melanoma tumor model, a combination of tumor-associated antigens (Trp1 214-237 and Trp2 173-196 ) was loaded into PSC7A NPs. The peptide-PSC7A NPs group also showed significantly improved tumor growth inhibition and extended survival time compared to the PBS control and peptide or PSC7A NPs alone groups (Figure 13B). In these two models, the PSC7A nanovaccine showed slightly improved tumor inhibition in the TC-1 model and a similar response in the B16F10 model compared to the PC7A nanovaccine at the same dose (Figure 14).

[0102] The results show that dUPS polymers bearing cyclic amines exhibit stronger STING binding affinity and interferon induction compared to those bearing dialkylamines, among which PSC7A is optimal. In vivo studies in two mouse tumor models show that antigen-loaded PSC7A NPs can effectively confer antitumor immunity by significantly improving tumor growth inhibition and animal survival.

[0103] Example 6: Evaluation of short-term and long-term safety of nanoparticles For polymers that actively engage the innate immune system, especially through the STING pathway, safety indications for repeated dosing during treatment are paramount. In this study, the dUPS PSC7A polymer used in vaccination studies was directly compared to its non-degradable PMMA-based predecessor, PC7A (Figure 15A). 6-8 week-old C57BL / 6 mice were subcutaneously injected with high doses of PSC7A NPs or PC7A NPs (300 μg, 10 times the vaccine dose) in the right flank. Serum was collected 24 hours after injection and systemic inflammatory cytokine concentrations were measured. No obvious acute renal or hepatotoxicity was observed 24 hours after treatment with either polymer (Figure 15B). Overall, systemic cytokine expression was more highly induced by PC7A NPs than by PSC7A NPs (Figure 15C), indicating a lesser systemic inflammatory response to PSC7A NPs. Histological analysis of the major organs (heart, liver, spleen, and kidneys) showed no significant changes following treatment with any of the polymers compared to PBS (Figure 16).

[0104] In long-term safety studies, the superiority of biodegradable PSC7A over non-degradable PC7A is more pronounced. In this assay, mice were subcutaneously injected with PBS, 300 μg PSC7A NPs, or 300 μg PC7A NPs and observed over a period of 60 days (Figure 17A). To monitor the progression, the surface area of ​​the resulting subcutaneous nodules was calculated based on an ellipse model (Figure 17B). Within 1 day after administration, a large acute inflammatory response was observed at the injection site for both PC7A and PSC7A, likely due to innate immune stimulation. Histologically, a large amount of neutrophil infiltration and necrotic debris was observed 24 hours after injection (day 1 time point, Figure 18). After this initial acute inflammatory response, the subcutaneous nodules decreased in size and gradually transitioned to a chronic granulomatous inflammatory response with more infiltration of macrophages and lymphocytes (days 15 and 30, Figure 18). PSC7A-induced nodules reduced in size at a faster rate than those induced by PC7A, indicating that the PSC7A polymer is degraded and expelled from the injection site, allowing eventual healing of the tissue. The half-life of PSC7A nodule size reduction was approximately 13 days, supporting the above chemical data on degradation rates. In contrast, PC7A-induced nodules reduced in size over time until 45 days after administration, after which the nodules remained constant in size and appearance. On day 60, skin tissues at the injection sites of all remaining mice were harvested for histological analysis (Figure 17C). Macroscopically, 6 / 6 skin samples from the PC7A group contain small, firm, yellow nodules. In contrast, none of the PSC7A groups (0 / 6) contain nodules and are similar in appearance to the PBS-treated group. H&E staining on day 60 reveals nodules surrounded by granulomatous inflammation with a "core / wall" appearance in mice treated with PC7A (Figure 17D). Here, the "wall" is composed mainly of macrophages with scattered lymphocytes and neutrophils resulting from acute and chronic innate irritation and foreign body reactions, while the "core" is necrotic and consists mainly of protein debris of dying cells with some infiltrating macrophages, neutrophils, and lymphocytes.In contrast, skin tissue from mice treated with PSC7A demonstrates complete disappearance of nodules and restoration to a healthy state by comparison with mice treated with PBS.

[0105] In vivo safety studies show that both PSC7A and PC7A induce a rapid innate inflammatory response over the short term, with systemic cytokine levels from PSC7A NPs being lower than PC7A NPs. Long-term PSC7A degradation allows complete healing at the injection site, whereas nodules surrounded by granulomatous inflammation persist at the PC7A site. Taken together, these data support the complete degradation of PSC7A over time, and a markedly improved safety profile compared to PC7A.

[0106] All compositions and methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described in terms of specific embodiments, it will be apparent to those skilled in the art that modifications may be applied to the compositions and methods, and to the steps or order of steps of the methods described herein, without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein, while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure, as defined by the appended claims.

[0107] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007676331000035.tif183128

Claims

1. formula: or a pharma- ceutically acceptable salt thereof, During the ceremony: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 40 to 180; z is an integer from 0 to 200; where either y or z monomers are randomly distributed in the polymer; X 1 , X 2 , X 1 ′, and X 2 ' are each independently O or NR a where: R a is an alkyl (C≦6) or substituted alkyl (C≦6) and R 2 and R 2 ' are each independently hydrogen, alkyl, (C≦8) , or substituted alkyl (C≦8) and R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and X 3 and X 3 ' are each independently O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and L and L′ are each independently a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 , Y 2 , Y 1 ′, and Y 2 Each ' is independently alkyl. (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 Or Y 1 ′ and Y 2 ' are combined and form alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, polymer.

2. below: or a pharma- ceutically acceptable salt thereof; Where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; p and q are each independently 1, 2, or 3; x is an integer from 10 to 200; y is an integer from 40 to 180; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of claim 1.

3. below: or a pharma- ceutically acceptable salt thereof; Where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; m is an integer from 1 to 8; x is an integer from 10 to 200; y is an integer from 40 to 180; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, 3. The polymer of claim 1 or claim 2.

4. below: or a pharma- ceutically acceptable salt thereof; Where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 40 to 180; X 1 and X 2 are each O or NR a where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and X 3 is O or NR b where: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of any one of claims 1 to 3.

5. below: or a pharma- ceutically acceptable salt thereof; Where: R 1 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or a thiol-reactive group; x is an integer from 10 to 200; y is an integer from 40 to 180; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and R 3 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of any one of claims 1 to 4.

6. below: or a pharma- ceutically acceptable salt thereof; Where: x is an integer from 10 to 200; y is an integer from 40 to 180; R 2 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) and L is a group of the formula: -X 4 -S(O) n -X 5 - During the ceremony: n is 0, 1, or 2; and X 4 and X 5 Each independently represents an alkanediyl (C≦8) or substituted alkanediyl (C≦8) and Y 1 and Y 2 are each independently an alkyl (C≦12) , substituted alkyl (C≦12) , alkenyl (C≦12) or substituted alkenyl (C≦12) or Y 1 and Y 2 are combined and alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substituted form of either group, The polymer of any one of claims 1 to 5.

7. 3. The polymer of claim 1 or claim 2, wherein p is 1.

8. 8. The polymer of claim 1, 2, or 7, wherein q is 1.

9. The polymer of any one of claims 1 to 3, 7, and 8, wherein m is 1, 2, or 3.

10. 10. The polymer of claim 9, wherein m is 2.

11. X 1 , X 2 , and X 3 The polymer of any one of claims 1-4 and 7-10, wherein

12. R 1 is alkyl (C≦8) or substituted alkyl (C≦8) The polymer of any one of claims 1 to 5 and 7 to 11, wherein

13. below:

13. The polymer of any one of claims 1 to 12, further defined as:

14. (A) a polymer according to any one of claims 1 to 13, and (B) Therapeutic Agent wherein the polymer encapsulates the therapeutic agent.

15. A pharmaceutical composition for treating a disease or disorder in a patient, comprising a therapeutically effective amount of the composition of claim 14, the therapeutic agent is sufficient to treat the disease or disorder, Pharmaceutical compositions.

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