Programmed immobilization of living cells using independent click pairs.

Therapeutic devices incorporating living cells or tissues have been intensively investigated for applications in tissue engineering and regenerative medicine. Because many biological processes are governed by spatially dependent signals, programmable immobilization of materials is crucial for manipulating multiple types of cells. In this study, click chemistry substrates were introduced onto the surfaces of cells and cover glass, and the cells were fixed on the cover glass via covalent bonds for selective cell deposition. Azide group (Az)-labeled living cells were prepared by metabolic labeling with azido sugars. Following the introduction of Az, TCO (trans-cyclooctene) was metabolically labeled into the living cells by reacting with TCO-DBCO (dibenzocyclooctyne). Az and TCO in the cells were detected using DBCO-FAM (fluorescein)and tetrazine-Cy3, respectively. The mixture of Az-labeled green fluorescent protein HeLa cells and TCO-labeled red fluorescent protein HeLa cells was reacted in a culture dish in which three different cover glasses, DBCO-, tetrazine-, or methyl-coated, were added. Az- or TCO-labeled cells could be immobilized in a functional group-dependent manner. Next, tetrazine-labeled cells were incubated on TCO- or Az-labeled cell layers instead of cover glass. Functional group-dependent immobilization was also achieved in the cell layer. Introducing substrates for the click reaction could achieve cell-selective immobilization on different patterned glass surfaces, as well as cell-cell immobilization.

Intracellular Delivery of Plasmid DNA Using Amphipathic Helical Cell-Penetrating Peptides Containing Dipropylglycine.

Cell-penetrating peptides (CPPs) serve as potent vehicles for delivering membrane-impermeable compounds, including nucleic acids, into cells. In a previous study, we reported the successful intracellular delivery of small interfering RNAs (siRNAs) with negligible cytotoxicity using a peptide containing an unnatural amino acid (dipropylglycine). In the present study, we employed the same seven peptides as the previous study to evaluate their efficacy in delivering plasmid DNA (pDNA) intracellularly. Although pDNA and siRNA are nucleic acids, they differ in size and biological function, which may influence the optimal peptide sequences for their delivery. Herein, three peptides demonstrated effective pDNA transfection abilities. Notably, only one of the three peptides previously exhibited efficient gene-silencing effect with siRNA. These findings validate our hypothesis and offer insights for the personalized design of CPPs for the delivery of pDNA and siRNA.

Fine tuning of the net charge alternation of polyzwitterion surfaced lipid nanoparticles to enhance cellular uptake and membrane fusion potential.

Lipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells.

The study presents the unique characteristics of small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) with different lengths of PGlu(DET-Car), revealing the length of PGlu(DET-Car) critically affects the formation of a stable LNP, the cellular uptake, membrane fusion, and gene silencing abilities.

Thermo-Responsive Polymer-siRNA Conjugates Enabling Artificial Control of Gene Silencing around Body Temperature.

PURPOSE: Controlling small interfering RNA (siRNA) activity by external stimuli is useful to exert a selective therapeutic effect at the target site. This study aims to develop a technology to control siRNA activity in a thermo-responsive manner, which can be utilized even at temperatures close to body temperature. METHODS: siRNA was conjugated with a thermo-responsive copolymer that was synthesized by copolymerization of N-isopropylacrylamide (NIPAAm) and hydrophilic N,N-dimethylacrylamide (DMAA) to permit thermally controlled interaction between siRNA and an intracellular gene silencing-related protein by utilizing the coil-to-globule phase transition of the copolymer. The composition of the copolymer was fine-tuned to obtain lower critical solution temperature (LCST) around body temperature, and the phase transition behavior was evaluated. The cellular uptake and gene silencing efficiency of the copolymer-siRNA conjugates were then investigated in cultured cells. RESULTS: The siRNA conjugated with the copolymer with LCST of 38.0°C exhibited ~ 11.5 nm of the hydrodynamic diameter at 37°C and ~ 9.8 nm of the diameter at 41°C, indicating the coil-globule transition above the LCST. In line with this LCST behavior, its cellular uptake and gene silencing efficiency were enhanced when the temperature was increased from 37°C to 41°C. CONCLUSION: By fine-tuning the LCST behavior of the copolymer that was conjugated with siRNA, siRNA activity could be controlled in a thermo-responsive manner around the body temperature. This technique may offer a promising approach to induce therapeutic effects of siRNA selectively in the target site even in the in vivo conditions.

Cancerous pH-responsive polycarboxybetaine-coated lipid nanoparticle for smart delivery of siRNA against subcutaneous tumor model in mice.

Lipid nanoparticles (LNPs) have been commonly used as a vehicle for nucleic acids, such as small interfering RNA (siRNA); the surface modification of LNPs is one of the determinants of their delivery efficiency especially in systemic administration. However, the applications of siRNA-encapsulated LNPs are limited due to a lack effective systems to deliver to solid tumors. Here, we report a smart surface modification using a charge-switchable ethylenediamine-based polycarboxybetaine for enhancing tumor accumulation via interaction with anionic tumorous tissue constituents due to selective switching to cationic charge in response to cancerous acidic pH. Our polycarboxybetaine-modified LNP could enhance cellular uptake in cancerous pH, resulting in facilitated endosomal escape and gene knockdown efficiency. After systemic administration, the polycarboxybetaine-modified LNP accomplished high tumor accumulation in SKOV3-luc and CT 26 subcutaneous tumor models. The siPLK-1-encapsulated LNP thereby accomplished significant tumor growth inhibition. This study demonstrates a promising potential of the pH-responsive polycarboxybetaine as a material for modifying the surface of LNPs for efficient nucleic acid delivery.

Iron chelation cancer therapy using hydrophilic block copolymers conjugated with deferoxamine.

Cancer cells have high iron requirements due to their rapid growth and proliferation. Iron depletion using iron chelators has a potential in cancer treatment. Previous studies have demonstrated that deferoxamine (DFO) specifically chelates Fe(III) and exhibited antitumor activity in clinical studies. However, its poor pharmacokinetics has limited the therapeutic potential and practical application. Although polymeric iron chelators have been developed to increase the blood retention, none of previous studies has demonstrated their potential in iron chelation cancer therapy. Here, we developed polymeric DFO by the covalent conjugation of DFO to poly(ethylene glycol)-poly(aspartic acid) (PEG-PAsp) block copolymers. The polymeric DFO exhibited iron-chelating ability comparable with free DFO, thereby arresting cell cycle and inducing apoptosis and antiproliferative activity. After intravenous administration, the polymeric DFO showed marked increase in blood retention and tumor accumulation in subcutaneous tumor models. Consequently, polymeric DFO showed significant suppression of the tumor growth compared with free DFO. This study reveals the first success of the design of polymeric DFO for enhancing iron chelation cancer therapy.

Sequential Self-Assembly Using Tannic Acid and Phenylboronic Acid-Modified Copolymers for Potential Protein Delivery.

Tannic acid (TA) can form stable complexes with proteins, attracting significant attention as protein delivery systems. However, its systemic application has been limited due to nonspecific interaction. Here, we report a simple technique to prepare systemically applicable protein delivery systems using sequential self-assembly of a protein, TA, and phenylboronic acid-conjugated PEG-poly(amino acid) block copolymers in aqueous solution. Mixing the protein and TA in aqueous solution led to covering of the protein with TA, and subsequent addition of the copolymer resulted in the formation of boronate esters between TA and copolymers, constructing the core-shell-type ternary complex. The ternary complex covered with PEG exhibited a small hydrodynamic diameter of ∼10-20 nm and prevented an unfavorable interaction with serum components, thereby accomplishing significantly prolonged blood circulation and enhanced tumor accumulation in a subcutaneous tumor model. The technique utilizing supramolecular self-assembly may serve as a novel approach for designing protein delivery systems.

Pyruvate Responsiveness Based on α-Oxohydrazone Formation for Intracellular siRNA Release from Polyion Complex-Based Carriers.

Selective release of small interfering RNA (siRNA) payloads in response to intracellular substances is a prerequisite for the smart design of siRNA carriers. In this context, we developed a molecular program that allows reactivity with pyruvate for siRNA release in the cell on the basis of polyionic-complex- (PIC-) based siRNA carriers. Hydrazide can react with the α-keto acid structure of anionic pyruvate to form α-oxohydrazone, resulting in the reduction of the cationic net charge of the cationic polymer bearing a hydrazide moiety, which in turn leads to an inefficient electrostatic interaction with anionic siRNA and the consequent destabilization of the PIC (i.e., PGlu [DET/hydrazide]) in pyruvate-enriched environments, such as the cytoplasm, thus achieving effective siRNA release from the PIC and its associated gene-silencing activity. The present study provides the rationale for an α-oxohydrazone-formation-based smart design of pyruvate-responsive materials in the cell.

Poly( N-isopropylacrylamide)-Based Polymer-Inducing Isothermal Hydrophilic-to-Hydrophobic Phase Transition via Detachment of Hydrophilic Acid-Labile Moiety.

The polymerization of N-isopropylacrylamide (NIPAAm) with ionizable monomers results in pH-responsive lower critical solution temperature (LCST) polymer which works in an ionization-dependent manner. However, gradual ionization of the comonomer occurs at a broad pH range due to the electrostatic field generated by the polymers, limiting the extent of LCST shift in response to pH change. Furthermore, excess introduction of comonomer may dull phase transition behavior. Here, we report the development of an ionization-independent LCST polymer that exerts a sharp isothermal hydrophilic-to-hydrophobic phase transition in response to slight pH change. Our polymer has a poly(NIPAAm/2-aminoisoprpylacrylamide (AIPAAm)) (P(NIPAAm/AIPAAm)) backbone that retains the continuous structural similarity of N-alkyl groups for preserving phase transition sensitivity, and primary amine for forming hydrophilic acid-labile 2-propionic-3-methylmaleic (PMM) amide linkage. The PMM moiety improves the polymer's hydrophilicity and drastically increases the LCST. Detachment of the PMM moiety in response to mild acidic condition (pH < 6.8) lowers the LCST to that of original P(NIPAAm/AIPAAm), permitting isothermal pH-responsive phase transition. Utilizing this mechanism, P(NIPAAm/AIPAAm) modified with PMM amide linkage exhibits a sharp hydrophilic-to-hydrophobic transition at a physiological temperature (37 °C) and, strikingly, facilitates interaction with cultured cells. Most importantly, our polymer showed significantly higher accumulation within a solid tumor after systemic injection compared to conventional PNIPAAm, which may be due to its phase transition responding to slightly acidic tumor microenvironment. Thus, this study provides a novel polymer that offers delicate control of LCST and pH-responsiveness suitable for use in even fuzzy biological environments.

An Ethylenediamine-based Switch to Render the Polyzwitterion Cationic at Tumorous pH for Effective Tumor Accumulation of Coated Nanomaterials.

Polyzwitterions are employed as coating polymers for biomaterials to induce an antifouling property on the surface. Fine-tuning the betaine structure switches the antifouling property to be interactive with anionic tissue constituents in response to a tumorous pH gradient. The ethylenediamine moiety in the carboxybetaine enabled stepwise protonation and initiated the di-protonation process around tumorous pH (6.5). The net charge of the developed polyzwitterion (PGlu(DET-Car)) was thus neutral at pH 7.4 for antifouling, but was cationic at pH 6.5 for interaction with anionic constituents. Quantum dots coated with PGlu(DET-Car) exhibited comparable stealth and enhanced tumor accumulation relative to the PEG system. The present study provides a novel design of smart switchable polyzwitterion based on a precise control of the net charge.

Artificial Control of Gene Silencing Activity Based on siRNA Conjugation with Polymeric Molecule Having Coil-Globule Transition Behavior.

A new strategy for controlling gene silencing activity of siRNA in the cell was developed in the present study. siRNA was linearly conjugated with PNIPAAm, where coil-globule transition of the conjugated PNIPAAm allows thermoresponsive exposure of the vicinal siRNA molecule; a coil form of PNIPAAm (T < LCST) inhibits siRNA interaction with gene silencing-related proteins due to the steric hindrance effect, while a globule form of PNIPAAm (T > LCST) allows a ready access of siRNA to gene silencing pathway. As a result, at T > LCST, PNIPAAm-siRNA elicited effective association of siRNA with a gene silencing-related protein of Ago2, while siRNA recruitment into the gene silencing pathway was significantly suppressed at T < LCST. Ultimately, gene silencing efficacy of PNIPAAm-siRNA was close to unconjugated siRNA at T > LCST (∼80%), while it was dramatically decreased to ∼20% at T < LCST, suggesting that coil-globule transition of the conjugated polymer can control the bioactivity of the vicinal siRNA molecule.

siRNA-Loaded Polyion Complex Micelle Decorated with Charge-Conversional Polymer Tuned to Undergo Stepwise Response to Intra-Tumoral and Intra-Endosomal pHs for Exerting Enhanced RNAi Efficacy.

Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.

Influence of RNA Strand Rigidity on Polyion Complex Formation with Block Catiomers.

Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association.

Fine-tuning of charge-conversion polymer structure for efficient endosomal escape of siRNA-loaded calcium phosphate hybrid micelles.

For efficient delivery of siRNA into the cytoplasm, a smart block copolymer of poly(ethylene glycol) and charge-conversion polymer (PEG-CCP) is developed by introducing 2-propionic-3-methylmaleic (PMM) amide as an anionic protective group into side chains of an endosome-disrupting cationic polyaspartamide derivative. The PMM amide moiety is highly susceptible to acid hydrolysis, generating the parent cationic polyaspartamide derivative at endosomal acidic pH 5.5 more rapidly than a previously synthesized cis-aconitic (ACO) amide control. The PMM-based polymer is successfully integrated into a calcium phosphate (CaP) nanoparticle with siRNA, constructing PEGylated hybrid micelles (PMM micelles) having a sub-100 nm size at extracellular neutral pH 7.4. Ultimately, PMM micelles achieve the significantly higher gene silencing efficiency in cultured cancer cells, compared to ACO control micelles, probably due to the efficient endosomal escape of the PMM micelles. Thus, it is demonstrated that fine-tuning of acid-labile structures in CCP improves the delivery performance of siRNA-loaded nanocarriers.

pH-responsive polyzwitterion covered nanocarriers for DNA delivery.

The success of gene therapy relies on gene nanocarriers to achieve therapeutic effects in vivo. Surface shielding of poly(ethylene glycol) (PEG), known as PEGylation, onto gene delivery carriers is a predominant strategy for extending blood circulation and improving therapeutic outcomes in vivo. Nevertheless, PEGylation frequently compromises the transfection efficiency by decreasing the interactions with the cellular membrane of the targeted cells, thereby preventing the cellular uptake and the subsequent endosomal escape. Herein, we developed a stepwise pH-responsive polyplex micelle for the plasmid DNA delivery with the surface covered by ethylenediamine-based polycarboxybetaines. This polyplex micelle switched its surface charge from neutral at pH 7.4 to positive at tumorous and endo-/lysosomal pH (i.e., pH 6.5 and 5.5, respectively), thus enhancing the cellular uptake and facilitating the endosomal escape toward efficient gene transfection. Additionally, the polyplex micelle demonstrated prolonged blood circulation as well as enhanced tumor accumulation, leading to highly effective tumor growth suppression by delivering an antiangiogenic gene. These results suggest the usefulness of a pH-responsive charge-switchable shell polymer on the surface of the polyplex micelle for the efficient nucleic acid delivery.

Polymeric ligands comprising sulfur-containing amino acids for targeting tumor-associated amino acid transporters.

Various cancer cells overexpress L-type amino acid transporter 1 (LAT1) to take up a large number of neutral amino acids such as phenylalanine and methionine, and LAT1 transporter should be a promising target for cancer diagnosis and therapy. However, only a few studies reported drug delivery systems targeting LAT1 probably due to limited knowledge about the interaction between LAT1 and its substrate. Here, we developed polymers having methionine (Met)- or cysteine (Cys)-like structures on their side chains to examine their affinity with LAT1. While both the Met- and Cys-modified polymers exhibited efficient cellular uptake selectively in cancer cells, the Met-modified polymers exhibited higher cellular uptake efficiency in an LAT1-selective manner than the Cys-modified polymers. In the in vivo study, the intraperitoneally injected Met-modified polymers showed appreciable tumor-selective accumulation in the peritoneal dissemination model, and importantly, Met-modified polymers conjugated with photosensitizers exhibited significant therapeutic effects upon photoirradiation with reduced photochemical damage to normal organs. Our results may provide important knowledge about the polymer-LAT1 interaction, and the Met-modified polymers should offer a new concept for designing LAT1-targeting drug delivery systems.

Accelerated polymer-polymer click conjugation by freeze-thaw treatment.

Herein, we report a unique technique to accelerate polymer-SNA conjugation based on copper-free click chemistry: gradual freeze-thawing of the reaction solution substantially increases the conjugation rate possibly because of the reactant concentration at the microenvironment scale. This technique was applied to the conjugation between a small interfering RNA (siRNA) and PEG in an aqueous buffer at/below room temperature.

Changeable net charge on nanoparticles facilitates intratumor accumulation and penetration.

The Enhanced Permeability and Retention (EPR) effect is a golden strategy for the nanoparticle (NP)-based targeting of solid tumors, and the surface property of NPs might be a determinant on their targeting efficiency. Poly(ethylene glycol) (PEG) is commonly used as a shell material; however, it has been pointed out that PEG-coated NPs may exhibit accumulation near tumor vasculature rather than having homogenous intratumor distribution. The PEG shell plays a pivotal role on prolonged blood circulation of NPs but potentially impairs the intratumor retention of NPs. In this study, we report on a shell material to enhance tumor-targeted delivery of NPs by maximizing the EPR effect: polyzwitterion based on ethylenediamine-based carboxybetaine [PGlu(DET-Car)], which shows the changeable net charge responding to surrounding pH. The net charge of PGlu(DET-Car), is neutral at physiological pH 7.4, allowing it to exhibit a stealth property during the blood circulation; however, it becomes cationic for tissue-interactive performance under tumorous acidic conditions owing to the stepwise protonation behavior of ethylenediamine. Indeed, the PGlu(DET-Car)-coated NPs (i.e., gold NPs in the present study) exhibited prolonged blood circulation and remarkably enhanced tumor accumulation and retention than PEG-coated NPs, achieving 32.1% of injected dose/g of tissue, which was 4.2 times larger relative to PEG-coated NPs. Interestingly, a considerable portion of PGlu(DET-Car)-coated NPs clearly penetrated into deeper tumor sites and realized the effective accumulation in hypoxic regions, probably because the cationic net charge of PGlu(DET-Car) is augmented in more acidic hypoxic regions. This study suggests that the changeable net charge on the NP surface in response to tumorous acidic conditions is a promising strategy for tumor-targeted delivery based on the EPR effect.

Sequentially Self-Assembled Nanoreactor Comprising Tannic Acid and Phenylboronic Acid-Conjugated Polymers Inducing Tumor-Selective Enzymatic Activity.

The construction of enzyme delivery systems, which can control enzymatic activity at a target site, is important for efficient enzyme-prodrug therapy/diagnosis. Herein we report a facile technique to construct a systemically applicable ß-galactosidase (ß-Gal)-loaded ternary complex comprising tannic acid (TA) and phenylboronic acid-conjugated polymers through sequential self-assembly in aqueous solution. At physiological conditions, the ternary complex exhibited a hydrodynamic diameter of ∼40 nm and protected the loaded ß-Gal from unfavorable degradation by proteinase. Upon cellular internalization, the ternary complex recovered ß-Gal activity by releasing the loaded ß-Gal. The intravenously injected ternary complex thereby delivered ß-Gal to the target tumor in a subcutaneous tumor model and exerted enhanced and selective enzymatic activity at the tumor site. Sequential self-assembly with TA and phenylboronic acid-conjugated polymers may offer a novel approach for enzyme-prodrug theragnosis.

Potential urinary monitoring of the enhanced permeability and retention effect using MMP-2-responsive poly(ethylene glycol) derivatives.

The enhanced permeability and retention (EPR) effect is fundamental to tumor-targeted drug delivery using nanoparticles. However, recent studies reported heterogeneity of the EPR effect, and companion diagnostics are considered to be key to predicting and optimizing the benefits of the EPR effect. Here, as a new material to simply endow the function of companion diagnostics to nanoparticles, we designed a poly(ethylene glycol) (PEG) derivative conjugated with low molecular fluorescent dye through synthetic substrate linker that can be cleaved in response to MMP-2, which is overexpressed in tumor extracellular matrix. Upon tumor accumulation, the low molecular fluorescent dye is released from the PEG and quickly excreted to urine, thereby reporting its tumor accumulation level as a fluorescent signal in the urine. In this study, this urinary reporter was conjugated with albumin, and the functionalized albumin exhibited efficient accumulation in various tumors. Importantly, the functionalized albumin exhibited significantly higher excretion of the fluorescent dye in the urine in mice with tumors compared with those without tumors. The PEG derivatives proposed in this study may be a promising tool to predict the EPR effect in individual cancer patients.