Magnetic DNA Vector Constructed from PDMAEMA Polycation and PEGylated Brush-Type Polyanion with Cross-Linkable Shell

A novel magnetic-responsive complex composed of polycation, DNA, and polyanion has been constructed via electrostatic interaction. The magnetic nanoparticles (MNPs) were first coated with a polycation, poly­[2-(dimethylamino)­ethyl methacrylate] end-capped with cholesterol moiety (Chol-PDMAEMA₃₀), and then binded with DNA through electrostatic interaction; the complexes were further interacted with the brush-type polyanion, namely poly­[poly­(ethylene glycol)­methyl ether methacrylate]-<i>block</i>-poly­[methacrylic acid carrying partial mercapto groups] (PPEGMA-<i>b</i>-PMAA_SH). The resulting magnetic particle/DNA/polyion complexes could be stabilized by oxidizing the mercapto groups to form cross-linking shell with bridging disulfide (S–S) between PPEGMA-<i>b</i>-PMAA_SH molecular chains. The interactions among DNA, Chol-PDMAEMA coated MNPs, and PPEGMA-<i>b</i>-PMAA_SH were studied by agarose gel retardation assay. The complexes were fully characterized by means of zeta potential, transmission electron microscopy (TEM), dynamic light scattering (DLS) measurements, cytotoxicity assay, antinonspecific protein adsorption, and <i>in vitro</i> transfection tests. All these results indicate that this kind of magnetic-responsive complex has potential applications for gene vector

Glucose-Sensitive Polyphosphoester Diblock Copolymer for an Insulin Delivery System

In this study, we report a diblock copolymer based on a polyphosphate backbone and pendant phenylboronic acid with glucose sensitivity. The copolymer, abbreviated as (PBYP-g-MPBA)-b-PEEP, was prepared via a combination of ring-opening copolymerization, “click” chemistry, and amide reaction, in which the PBYP and PEEP blocks, respectively, represent two kinds of polyphosphoester structures and MPBA represents 3-mercaptopropionic acid modified with 3-aminophenylboronic acid. The amphiphilic copolymer (PBYP-g-MPBA)-b-PEEP could self-assemble into core–shell nanoparticles (NPs) in aqueous solutions. The average particle size and morphology of the NPs were measured by dynamic light scattering and transmission electron microscopy, respectively. The phenomenon that the NPs swelled at different glucose concentrations is due to the formation of boronate esters between the diol groups of glucose and boronic acid groups of phenylboronic acid. Fluorescein isothiocyanate (FITC)–insulin was loaded into the NPs and triggered to release in the presence of glucose. The more the glucose in the release media, the more the FITC–insulin released and the faster the release rate. Methyl thiazolyl tetrazolium assays and hemolysis tests proved that the (PBYP-g-MPBA)-b-PEEP copolymers had good biocompatibility. All of these results verify that the glucose-sensitive polyphosphoester diblock copolymer is highly promising for an insulin delivery system

PolymerDoxorubicin Prodrug with Biocompatibility pH Response, and Main Chain Breakability Prepared by Catalyst-Free Click Reaction

Click chemistry has increasing applications of the development of polymer materials and modification of drug carriers. The amino–yne click polymerization reacts quickly at room temperature without catalyst, and the enamine bond (-ena-) gained from the reaction is sensitive to acid and can be used to prepare stimulus-responsive polymeric prodrugs. Herein, we report an alkynyl-terminated polymer containing alternately distributed low molecular weight polyethylene glycol (PEG) and hexamethylenediamino (HMDA) linked by enamine bonds, abbreviated as A-P­(PEG-alt-HMDA)-A, which was synthesized within 3 h at 35 °C without catalyst. The polymer was verified to have good water solubility, biocompatibility, and acid-sensitive fracturing. Then, a pH-responsive polymeric prodrug (DOX-ena-PPEG-ena-DOX) was further prepared through the amino–yne click reaction between the alkynyl groups of A-P­(PEG-alt-HMDA)-A and the amino group of doxorubicin hydrochloride (DOX·HCl). The resulting prodrug can self-assemble into nanoparticles (NPs) in aqueous solution. The pH responsiveness of the prodrug NPs was demonstrated by a stability experiment of NPs and in vitro drug release behavior measurement. The accumulative release of doxorubicin (DOX) was tested with different pH media, which confirmed that the prodrug NPs could effectively dissociate and release drug under a weak acid microenvironment of lysosome/endosome. Subsequently, we investigated cell cytotoxicity and intracellular uptake of the prodrug. It turned out that the prodrug nanoparticles could be internalized into HeLa cells, release original DOX, and efficiently inhibit the proliferation of cancer cells. These results show that the pH-responsive DOX-ena-PPEG-ena-DOX has the potential for use in cancer therapy

Dual-Responsive Polyphosphoester-Doxorubicin Prodrug Containing a Diselenide Bond: Synthesis, Characterization, and Drug Delivery

The development of novel stimuli-responsive and biodegradable polyphosphoester-anticancer prodrugs is of importance in designing water-soluble prodrugs utilized in the field of drug delivery. In this study, the focus is on the synthesis of biocompatible and biodegradable diselenide-containing polyphosphoester [PEEP-b-PBYP-Se]2 using reduction-responsive di­(1-hydroxylundecyl) diselenide as an initiator to polymerize 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (BYP) and 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EOP). After that, a doxorubicin (DOX) derivative containing an azide group was linked onto the side chain of [PEEP-b-PBYP-Se]2 via the Cu­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) “click” reaction to yield a pH/reduction-responsive polymeric prodrug, namely [PEEP-b-(PBYP-hyd-DOX)-Se]2. The chemical structures of various polymers were characterized by nuclear magnetic resonance spectroscopy, ultraviolet–visible spectrophotometer, Fourier transform infrared spectroscopy, and high-performance liquid chromatography. The self-assembly behavior measured by dynamic light scattering and transmission electron microscopy clearly supported the formation of the prodrug nanoparticles (NPs). The results indicated that the polymeric prodrug NPs were relatively uniform spheres that could maintain stability in a physiological condition but be cleaved in acidic or reductive medium. Furthermore, the pH- and reduction-responsive properties of the prodrug NPs were investigated via drug release in vitro in different media. It turned out that the drug was efficiently released in acidic or reductive medium compared with that under physiological conditions. The results of methyl thiazolyl tetrazolium assays confirmed the favorable biocompatibility of [PEEP-b-PBYP-Se]2. Moreover, the cell cytotoxicity and intracellular uptake experiments were carried out to verify the efficient cellular proliferation inhibition. This finding contributes to the design of a novel diselenide-containing polyphosphoester-doxorubicin prodrug

Folate-Conjugated Polyphosphoester with Reversible Cross-Linkage and Reduction Sensitivity for Drug Delivery

To improve the therapeutic efficacy and circulation stability in vivo, we synthesized a new kind of drug delivery carrier based on folic acid conjugated polyphosphoester via the combined reactions of Michael addition polymerization and esterification. The produced amphiphilic polymer, abbreviated as P­(EAEP-AP)-LA-FA, could self-assemble into nanoparticles (NPs) with core-shell structure in water and reversible core cross-linked by lipoyl groups. Using the core cross-linked FA-conjugated nanoparticles (CCL-FA NPs) to encapsulate hydrophobic anticancer drug doxorubicin (DOX), we studied the stability of NPs, in vitro drug release, cellular uptake, and targeting intracellular release compared with both un-cross-linked FA-conjugated nanoparticles (UCL-FA NPs) and core cross-linked nanoparticles without FA conjugation (CCL NPs). The results showed that under the condition of pH 7.4, the DOX-loaded CCL-FA NPs could maintain stable over 72 h, and only a little DOX release (∼15%) was observed. However, under the reductive condition (pH 7.4 containing 10 mM GSH), the disulfide-cross-linked core would be broken up and resulted in 90% of DOX release at the same incubation period. The study of methyl thiazolyl tetrazolium (MTT) assay indicated that the DOX-loaded CCL-FA NPs exhibited higher cytotoxicity (IC₅₀: 0.33 mg L^–1) against HeLa cells than the DOX-loaded CCL NPs without FA. These results indicate that the core cross-linked FA-conjugated nanoparticles have unique stability and targetability

Functional cRGD-Conjugated Polymer Prodrug for Targeted Drug Delivery to Liver Cancer Cells

To overcome the limitation of conventional nanodrugs in tumor targeting efficiency, coupling targeting ligands to polymeric nanoparticles can enhance the specific binding of nanodrugs to tumors. Cyclo­(Arg-Gly-Asp-d-Phe-Lys) (abbreviated as c­(RGDfK)) peptide has been widely adopted due to its high affinity to the tumor marker αvβ3 integrin receptor. In this study, we develop a cRGD peptide-conjugated camptothecin (CPT) prodrug, which enables self-assembly of nanoparticles for precise targeting and enrichment in tumor tissue. We first synthesized a camptothecin derivative (CPT-ss-N3) with a reduction-sensitive bond and simultaneously modified PEG to obtain cRGD-PEG-N3. After ring-opening polymerization of the 2-(but-3-yn-1-yolxy)-2-oxo-1,3,2-dioxaphospholane (BYP), an amphiphilic polymeric prodrug, referred to as cRGD-PEG-g-(PBYP-ss-CPT), was obtained via copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. The self-assembly in buffer solution of the cRGD-functional prodrug was studied through DLS and TEM. The in vitro drug release behavior of cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles was investigated. The results show that the nanoparticles are reduction-responsive and the bonded CPT can be released. Endocytosis and MTT assays demonstrate that the cRGD-conjugated prodrug has better affinity for tumor cells, accumulates more intracellularly, and is therefore, more effective. The in vivo drug metabolism studies show that nanoparticles greatly prolong the retention time in circulation. By monitoring drug distribution in tumor and in various tissues, we find that free CPT can be rapidly metabolized, resulting in low accumulation in all tissues. However, cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles accumulate in tumor tissues in higher amounts than PEG-g-(PBYP-ss-CPT) nanoparticles, except for the inevitable capture by the liver. This indicates that the nanomedicine with cRGD has a certain targeting property, which can improve drug delivery efficiency

Functional cRGD-Conjugated Polymer Prodrug for Targeted Drug Delivery to Liver Cancer Cells

To overcome the limitation of conventional nanodrugs in tumor targeting efficiency, coupling targeting ligands to polymeric nanoparticles can enhance the specific binding of nanodrugs to tumors. Cyclo­(Arg-Gly-Asp-d-Phe-Lys) (abbreviated as c­(RGDfK)) peptide has been widely adopted due to its high affinity to the tumor marker αvβ3 integrin receptor. In this study, we develop a cRGD peptide-conjugated camptothecin (CPT) prodrug, which enables self-assembly of nanoparticles for precise targeting and enrichment in tumor tissue. We first synthesized a camptothecin derivative (CPT-ss-N3) with a reduction-sensitive bond and simultaneously modified PEG to obtain cRGD-PEG-N3. After ring-opening polymerization of the 2-(but-3-yn-1-yolxy)-2-oxo-1,3,2-dioxaphospholane (BYP), an amphiphilic polymeric prodrug, referred to as cRGD-PEG-g-(PBYP-ss-CPT), was obtained via copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. The self-assembly in buffer solution of the cRGD-functional prodrug was studied through DLS and TEM. The in vitro drug release behavior of cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles was investigated. The results show that the nanoparticles are reduction-responsive and the bonded CPT can be released. Endocytosis and MTT assays demonstrate that the cRGD-conjugated prodrug has better affinity for tumor cells, accumulates more intracellularly, and is therefore, more effective. The in vivo drug metabolism studies show that nanoparticles greatly prolong the retention time in circulation. By monitoring drug distribution in tumor and in various tissues, we find that free CPT can be rapidly metabolized, resulting in low accumulation in all tissues. However, cRGD-PEG-g-(PBYP-ss-CPT) nanoparticles accumulate in tumor tissues in higher amounts than PEG-g-(PBYP-ss-CPT) nanoparticles, except for the inevitable capture by the liver. This indicates that the nanomedicine with cRGD has a certain targeting property, which can improve drug delivery efficiency

Efficient Click Synthesis of a Protonized and Reduction-Sensitive Amphiphilic Small-Molecule Prodrug Containing Camptothecin and Gemcitabine for a Drug Self-Delivery System

Drug self-delivery systems consisting of small-molecule active drugs with nanoscale features for intracellular delivery without the need for additional polymeric carriers have drawn much attention recently. In this work, we proposed a highly efficient strategy to fabricate protonized and reduction-responsive self-assembled drug nanoparticles from an amphiphilic small-molecule camptothecin–ss-1,2,3-triazole–gemcitabine conjugate (abbreviated as CPT–ss-triazole–GEM) for combination chemotherapy, which was prepared via a Cu­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) “click” reaction. To obtain this drug–triazole–drug conjugate, we first prepared a CPT derivate containing a propargyl group linked with a disulfide group and a GEM derivate attached to an azide group. Subsequently, the two kinds of modified drugs were connected together through a CuAAC reaction between the alkynyl and azide groups to yield the CPT–ss-triazole–GEM prodrug. The characterizations of chemical structures of these intermediates and the final product were performed by 1H NMR, Fourier transform infrared, and liquid chromatography/mass spectrometry measurements. This amphiphilic small-molecule drug–triazole–drug conjugate displayed a high drug loading content, that is, 36.0% of CPT and 27.2% of GEM. This kind of amphiphilic small-molecule prodrugs could form spherical nanoparticles in an aqueous solution in the absence of any other polymeric carriers, in which the hydrophobic CPT formed the core of the nanoparticles, whereas the hydrophilic GEM and protonated 1,2,3-triazole group yielded the shell. In the tumor microenvironment, the prodrug nanoparticles could release both pristine drugs simultaneously. Under the conditions of pH 7.4, and pH 7.4 and 2 μM glutathione (GSH), the prodrug nanoparticles could maintain stability and only 7% of CPT was leaked. However, in a high-GSH environment (pH 7.4 and 10 mM GSH) with the same incubation time, the disulfide linkage would be dissociated and lead to about 34% of CPT release. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test demonstrated that these prodrug nanoparticles showed a higher cytotoxicity toward HepG2 cells than free CPT and free GEM on both 48 and 72 h of incubation. Both in vitro cellular uptake and flow cytometry results implied that these prodrug nanoparticles could be internalized by HepG2 cells with efficient drug release inside cells. The pharmacokinetics and tissue distribution of the prodrug showed a moderate half-life in vivo, and the prodrug peak concentration in most of the collected tissues appeared at 0.25 h after administration. In addition, the CPT–ss-triazole–GEM prodrug could not cross the blood–brain barrier. Even more important is the fact that there is no accumulation in tissues and a rapid elimination of this small-molecule prodrug could be achieved. In brief, this protonized and reduction-sensitive prodrug simultaneously binds both antitumor drugs and has good self-delivery behavior through the donor–acceptor interaction of the H-bonding ligand, that is, the 1,2,3-triazole group. It provides a new method for combined drug therapy

One-Pot Synthesis of pH/Redox Responsive Polymeric Prodrug and Fabrication of Shell Cross-Linked Prodrug Micelles for Antitumor Drug Transportation

Shell cross-linked (SCL) polymeric prodrug micelles have the advantages of good blood circulation stability and high drug content. Herein, we report on a new kind of pH/redox responsive dynamic covalent SCL micelle, which was fabricated by self-assembly of a multifunctional polymeric prodrug. At first, a macroinitiator PBYP-<i>ss</i>-<i>i</i>BuBr was prepared via ring-opening polymerization (ROP), wherein PBYP represents poly­[2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane]. Subsequently, PBYP-<i>hyd</i>-DOX-<i>ss</i>-P­(DMAEMA-<i>co</i>-FBEMA) prodrug was synthesized by a one-pot method with a combination of atom transfer radical polymerization (ATRP) and a Cu­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction using a doxorubicin (DOX) derivative containing an azide group to react with the alkynyl group of the side chain in the PBYP block, while DMAEMA and FBEMA are the abbriviations of <i>N</i>,<i>N</i>-(2-dimethylamino)­ethyl methacrylate and 2-(4-formylbenzoyloxy)­ethyl methacrylate, respectively. The chemical structures of the polymer precursors and the prodrugs have been fully characterized. The SCL prodrug micelles were obtained by self-assembly of the prodrug and adding cross-linker dithiol bis­(propanoic dihydrazide) (DTP). Compared with the shell un-cross-linked prodrug micelles, the SCL prodrug micelles can enhance the stability and prevent the drug from leaking in the body during blood circulation. The average size and morphology of the SCL prodrug micelles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The SCL micelles can be dissociated under a moderately acidic and/or reductive microenvironment, that is, endosomal/lysosomal pH medium or high GSH level in the tumorous cytosol. The results of DOX release also confirmed that the SCL prodrug micelles possessed pH/reduction responsive properties. Cytotoxicity and cellular uptake analyses further revealed that the SCL prodrug micelles could be rapidly internalized into tumor cells through endocytosis and efficiently release DOX into the HeLa and HepG2 cells, which could efficiently inhibit the cell proliferation. This study provides a fast and precise synthesis method for preparing multifunctional polymer prodrugs, which hold great potential for optimal antitumor therapy

Glucose-Sensitive Core-Cross-Linked Nanoparticles Constructed with Polyphosphoester Diblock Copolymer for Controlling Insulin Delivery

This work aims to construct biocompatible, biodegradable core-cross-linked and insulin-loaded nanoparticles which are sensitive to glucose and release insulin via cleavage of the nanoparticles in a high-concentration blood glucose environment. First, a polyphosphoester-based diblock copolymer (PBYP-g-Gluc)-b-PEEP was prepared via ring-opening copolymerization (ROP) and the copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) in which PBYP and PEEP represent the polymer segments from 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane and 2-ethoxy-2-oxo-1,3,2-dioxaphospholane, respectively, and Gluc comes from 2-azidoethyl-β-d-glucopyranoside (Gluc-N3) that grafted with PBYP. The structure and molecular weight of the copolymer were characterized by 1H NMR, 31P NMR, GPC, FT-IR, and UV–vis measurements. The amphiphilic copolymer could self-assemble into core–shell uncore-cross-linked nanoparticles (UCCL NPs) in aqueous solutions and form core-cross-linked nanoparticles (CCL NPs) after adding cross-linking agent adipoylamidophenylboronic acid (AAPBA). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to study the self-assembly behavior of the two kinds of NPs and the effect of different Gluc group contents on the size of NPs further to verify the stability and glucose sensitivity of CCL NPs. The ability of NPs to load fluorescein isothiocyanate-labeled insulin (FITC–insulin) and their glucose-triggered release behavior were detected by a fluorescence spectrophotometer. The results of methyl thiazolyl tetrazolium (MTT) assay and hemolysis activity experiments showed that the CCL NPs had good biocompatibility. An in vivo hypoglycemic study has shown that FITC–insulin-loaded CCL NPs could reduce blood glucose and have a protective effect on hypoglycemia. This research provides a new method for constructing biodegradable and glucose-sensitive core-cross-linked nanomedicine carriers for controlled insulin release