Charge‐Conversional Polyionic Complex Micelles—Efficient Nanocarriers for Protein Delivery into Cytoplasm

Special delivery! Polyionic complex (PIC) micelles that contain the charge‐conversional moieties citaconic amide or cis‐aconitic amide were developed for cytoplasmic protein delivery. The increase of the charge density on the protein cargo helped the stability of the PIC micelles without cross‐linking, and the charge‐conversion in endosomes induced the dissociation of the PIC micelles to result in efficient endosomal release (see picture).

     

pH‐Triggered Charge‐Reversal Polyurethane Micelles for Controlled Release of Doxorubicin

A series of pH‐triggered charge‐reversal polyurethane copolymers (PS‐PUs) containing methoxyl‐poly(ethylene glycol) (mPEG), carboxylic acid groups, and piperazine groups is presented in this work. The obtained PS‐PUs copolymers can form into stable micelles at pH 7.4, which response to a narrow pH change (5.5–7.5) and show a tunable pH‐triggered charge‐reversal property. Doxorubicin (DOX) is encapsulated into the PS‐PU micelles as a model drug. The drug release of DOX‐loaded PS‐PU micelles shows an obviously stepped‐up with reducing the pH. Meanwhile, it is found that the charge‐reversal property can improve the cellular uptake behavior and intracellular drug release in both HeLa cells and MCF‐7 cells. Additionally, the time‐dependent cytotoxicity of the DOX‐loaded PS‐PU micelles is confirmed by MTT assay. Attributed to the tunable charge‐reversal property through changing the molar ratio of piperazine/carboxyl, the PS‐PU micelles will be a potential candidate as an intelligent drug delivery system in future studies.

     

Tumor‐Acidity Activated Surface Charge‐Conversion of Polymeric Nanocarriers for Enhanced Cell Adhesion and Targeted Drug Release

The development of stimuli‐responsive polymeric nanocarriers could significantly enhance drug bioavailability due to improved pharmacokinetics and biodistribution. However, in the drug delivery process, the poor cell uptake of drug‐loaded carriers has greatly limited the therapeutic efficiency for anti‐cancer applications. Herein, 2,3‐dimethylmaleic anhydride (DMMA) is engineered into the well‐defined biodegradable amphiphilic block copolymer poly(D,L‐lactide)‐block‐poly(2‐aminoethyl methacrylate) (PLA‐b‐PAEMA) to construct a tumor‐acidity activated nanocarrier (PLA‐b‐PAEMA/DMMA) for potential tumor therapy. After the loading of positively charged DOX·HCl into the negatively charged corona structure through electrostatic attraction, this carrier is expected to prolong the blood circulation time and smartly convert surface charge from negative to positive for enhanced tumor cell uptake and targeted drug release. Furthermore, this carrier exhibits additional cytotoxicity for tumor cells after the tumor‐acidity activated surface charge‐conversion from negative to positive. Thus, this smart carrier is a feasible candidate for potential cancer therapy.

     

Facile Fabrication of Thermo‐Responsive and Reduction‐Sensitive Polymeric Micelles for Anticancer Drug Delivery

The development of thermo‐responsive and reduction‐sensitive polymeric micelles based on an amphiphilic block copolymer poly[(PEG‐MEMA)‐co‐(Boc‐Cyst‐MMAm)]‐block‐PEG (denoted PEG‐P‐SS‐HP) for the intracellular delivery of anticancer drugs is reported. PTX, as model drug, was loaded into the PEG‐P‐SS‐HP micelles with an encapsulation efficiency >90%, resulting in a high drug loading content (up to 35 wt%). The PTX‐loaded PEG‐P‐SS‐HP micelles show slow drug release in PBS and rapid release after incubation with DTT. The PTX‐loaded micelles display a better cytotoxic effect than the free drug, whereas empty micelles are found to be non‐toxic. The thermo‐responsive and reduction‐sensitive polymeric micelles described may serve as promising carriers for cytostatic drugs.

     

Inside Cover: Charge‐Conversional Polyionic Complex Micelles—Efficient Nanocarriers for Protein Delivery into Cytoplasm (Angew. Chem. Int. Ed. 29/2009)

A highly efficient protein delivery into cytoplasm is described by K. Kataoka, and co‐workers on page 5309 ff. The charge‐density increase of a protein cargo by reversible modification, which was based on the charge‐conversional moieties citaconic amide and cis‐aconitic amide, helped the stability of protein/block copolymer polyionic complex (PIC) micelles. The rapid protein charge conversion in endosomes induced the dissociation of the PIC micelles and efficient endosomal escape.

     

In Vitro Drug and Gene Delivery Using Random Cationic Copolymers Forming Stable and pH‐Sensitive Polymersomes

Stimuli‐sensitive polymeric vesicles or polymersomes as self‐assembled colloidal nanocarriers have received paramount importance for their integral role as delivery system for therapeutics and biotherapeutics. This work describes spontaneous polymersome formation at pH 7, as evidenced by surface tension, steady state fluorescence, dynamic light scattering, and microscopic studies, by three hydrophilic random cationic copolymers synthesized using N ,N‐(dimethylamino)ethyl methacrylate (DMAEM) and methoxy poly(ethylene glycol) monomethacrylate in different mole ratios. The results suggest that methoxy poly(ethylene glycol) chains constitute the bilayer membrane of the polymersomes and DMAEM projects toward water constituting the positively charged surface. The polymersomes have been observed to release their encapsulated guest at acidic pH as a result of transformation into polymeric micelles. All these highly biocompatible cationic polymers show successful gene transfection ability as nonviral vector on human cell line with different potential. Thus these polymers prove their utility as a potential delivery system for hydrophilic model drug as well as genetic material.

     

Synthesis and Characterization of pH‐Sensitive Positive‐charge Silica Nanoparticles for Oral Anionic Drug Delivery

The objective of this study is to utilize the pH sensitivity of modified mesoporous silica nanoparticles (MSN) for oral drug delivery. In the first time, a pH‐sensitive ionic liquid was synthesized through the quaternization of 3‐aminopropyltrimethoxysilane (3‐ATMS) with sodium monochloroacetate (SMCA). Then, silica nanoparticle was modified by this pH‐sensitive ionic liquid and converted to a pH‐sensitive positive‐charge silica nanoparticle (PCSN). The nanoparticle was characterized by FTIR and SEM. Naproxen as anionic drug molecules was entrapped in this pH‐sensitive positive‐charge silica nanoparticles (PCSN) and the in vitro release profiles were established separately in both (SGF, pH 1) and (SIF, pH 7.4).     

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH‐Cleavable Covalent Bonding for In Vivo Delivery

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half‐life, and adverse immune responses. Thus, protein delivery approaches based on stimuli‐responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH‐cleavable covalent bonding between proteins and block copolymers directed to pH‐triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH‐sensitive moiety, since the CDM? amide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)‐poly(l ‐lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin‐loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half‐life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.     

Nucleobase‐Functionalized Supramolecular Micelles with Tunable Physical Properties for Efficient Controlled Drug Release

Complementary nucleobase‐functionalized polymeric micelles, a combination of adenine‐thymine (A‐U) base pairs and a blend of hydrophilic–hydrophobic polymer pairs, can be used to construct 3D supramolecular polymer networks; these micelles exhibit excellent self‐assembly ability in aqueous solution, rapid pH‐responsiveness, high drug loading capacity, and triggerable drug release. In this study, a multi‐uracil functionalized poly(ε‐caprolactone) (U‐PCL) and adenine end‐capped difunctional oligomeric poly(ethylene glycol) (BA‐PEG) are successfully developed and show high affinity and specific recognition in solution owing to dynamically reversible A‐U‐induced formation of physical cross‐links. The U‐PCL/BA‐PEG blend system produces supramolecular micelles that can be readily adjusted to provide the desired critical micellization concentration, particle size, and stability. Importantly, in vitro release studies show that doxorubicin (DOX)‐loaded micelles exhibit excellent DOX‐encapsulated stability under physiological conditions. When the pH value of the solution is reduced from 7.4 to 5.0, DOX‐loaded micelles can be rapidly triggered to release encapsulated DOX, suggesting these polymeric micelles represent promising candidate pH‐responsive nanocarriers for controlled‐release drug delivery and pharmaceutical applications.

     

Contractive Polymeric Complex Micelles as Thermo‐Sensitive Nanopumps

Complex micelles were obtained from PS‐b‐PNIPAM‐b‐PAA micelles and PEG‐b‐P4VP block copolymers via the strong electrostatic interaction and hydrogen bonding between PAA and P4VP blocks in water. The PS block formed the core and the PAA/P4VP complex shell functioned as a semi‐permeable membrane which could control the permeation of small molecules. Between the core and shell, the large fluid‐filled space that was formed with the thermoresponsive PNIPAM gel could retain the loaded drug for a long period of time. With increasing temperature, the shrinkage of the PNIPAM coils pumped the drug out of the complex micelles. The complex micelles functioned as a contractive “nanopump”, which could potentially be applied as a thermosensitive controlled release system.

     

Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery

An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio‐reducible disulfide bond linked siRNA‐cell penetrating peptides (CPPs) conjugate is developed to suppress c‐myc gene expression of breast cancer (MCF‐7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA‐CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo.

     

Synthesis and Evaluation of Biocompatible pH‐Sensitive Hydrogels as Colon‐Specific Drug Delivery Systems

Because of the growing importance of pH‐sensitive hydrogels as drug delivery systems, biocompatible copolymeric hydrogels based N‐vinyl‐2‐pyrrolidinone (NVP) and methacrylic acid (MAA) were designed and synthesized. These hydrogels were investigated for oral drug delivery. Radical copolymerizations of N‐vinyl‐2‐pyrrolidinone (NVP) and methacrylic acid (MAA) with the various ratios of cross‐linking agent were carried out at 70 °C. Azabisisobutyronitrile (AIBN) was the free‐radical initiator employed and Cubane‐1,4‐dicarboxylic acid (CDA) linked to two 2‐hydroxyethyl methacrylate (HEMA) group was the crosslinking agent (CA) used for hydrogel preparations. The hydrogels were characterized by differential scanning calorimetry and FT‐IR. Equilibrium swelling studies were carried out in enzyme‐free simulated gastric and intestinal fluids (SGF and SIF, respectively). A model drug, olsalazine [3,3′‐azobis (6‐hydroxy benzoic acid)] (OSZ) as an azo derivative of 5‐aminosalicylic acid (5‐ASA), was entrapped in these gels and the in‐vitro release profiles were established separately in both enzyme‐free SGF and SIF. The drug‐release profiles indicated that the amount of drug released depended on the degree of swelling. The swelling was modulated by the amount of crosslinking of the polymer bonded drug (PBDs) prepared. Based on the great difference in hydrolysis rates at pH 1 and 7.4, these pH‐sensitive hydrogels appear to be good candidates for colon‐specific drug delivery.     

pH‐Responsive Drug‐Delivery Systems

In many biomedical applications, drugs need to be delivered in response to the pH value in the body. In fact, it is desirable if the drugs can be administered in a controlled manner that precisely matches physiological needs at targeted sites and at predetermined release rates for predefined periods of time. Different organs, tissues, and cellular compartments have different pH values, which makes the pH value a suitable stimulus for controlled drug release. pH‐Responsive drug‐delivery systems have attracted more and more interest as “smart” drug‐delivery systems for overcoming the shortcomings of conventional drug formulations because they are able to deliver drugs in a controlled manner at a specific site and time, which results in high therapeutic efficacy. This focus review is not intended to offer a comprehensive review on the research devoted to pH‐responsive drug‐delivery systems; instead, it presents some recent progress obtained for pH‐responsive drug‐delivery systems and future perspectives. There are a large number of publications available on this topic, but only a selection of examples will be discussed.     

Cell–Membrane Penetration of Tat‐Conjugated Polymeric Micelles: Effect of Tat Coating Density

Considerable efforts have been devoted to enhancing the cell penetration of nanoparticles by coating cell‐penetrating peptides (CPPs) on the surface. However, the internalization mechanism for a CPP at different concentrations varies a lot. It is acknowledged that the mechanism is restricted to endocytic pathway at relatively low concentrations; however, direct translocation becomes dominant at high concentrations. This raises an interesting question on how the surface Tat coating density of the nanoparticles would influence their cell–membrane interaction and the consequent internalization behavior. This study systematically investigates the effect of Tat peptides on the internalization behavior of polymeric micelles by tuning surface Tat coating density, incubation concentrations, incubation time, and other factors using poly(ethylene glycol)–poly(ε‐caprolactone) copolymer (PEG‐PCL) micelles. It is found that both energy‐dependent and energy‐independent pathways are involved in the cellular uptake process, and the Tat‐conjugated polymeric micelles strongly accumulated on the cell surface at initial stage. The membrane‐anchoring and internalization rate increase with the increasing Tat coating density. Furthermore, the increasing of Tat coating density accelerates the energy‐independent pathway. It is envisioned that this finding will further shed light on the surface modification of nanoparticles for enhanced cell penetration and direct translocation into cell cytoplasm.