Optimized Carbohydrate-Based Nanogel Formulation to Sensitize Hypoxic Tumors.

Solid tumors are often poorly vascularized, which impairs oxygen supply and drug delivery to the cells. This often leads to genetic and translational adaptations that promote tumor progression, invasion, metastasis, and resistance to conventional chemo-/radiotherapy and immunotherapy. A hypoxia-directed nanosensitizer formulation of a hypoxia-activated prodrug (HAP) was developed by encapsulating iodoazomycin arabinofuranoside (IAZA), a 2-nitroimidazole nucleoside-based HAP, in a functionally modified carbohydrate-based nanogel, facilitating delivery and accrual selectively in the hypoxic head and neck and prostate cancer cells. Although IAZA has been reported as a clinically validated hypoxia diagnostic agent, recent studies have pointed to its promising hypoxia-selective anti-tumor properties, which make IAZA an excellent candidate for further exploration as a multimodal theranostic of hypoxic tumors. The nanogels are composed of a galactose-based shell with an inner core of thermoresponsive (di(ethylene glycol) methyl ethyl methacrylate) (DEGMA). Optimization of the nanogels led to high IAZA-loading capacity (≅80-88%) and a slow time-controlled release over 50 h. Furthermore, nanoIAZA (encapsulated IAZA) displayed superior in vitro hypoxia-selective cytotoxicity and radiosensitization in comparison to free IAZA in the head and neck (FaDu) and prostate (PC3) cancer cell lines. The acute systemic toxicity profile of the nanogel (NG1) was studied in immunocompromised mice, indicating no signs of toxicity. Additionally, growth inhibition of subcutaneous FaDu xenograft tumors was observed with nanoIAZA, demonstrating that this nanoformulation offers a significant improvement in tumor regression and overall survival compared to the control.

Glycopolymer-Cell-Penetrating Peptide (CPP) Conjugates for Efficient Epidermal Growth Factor Receptor (EGFR) Silencing.

Overexpression of epidermal growth factor receptor (EGFR) is observed in multiple cancers such as colorectal, lung, and cervical solid tumors. Regulating the EGFR expression is an efficient strategy to manage these malignancies, and it can be achieved by using short interfering RNA (siRNA). Cell-penetrating peptides (CPPs) demonstrated an excellent capability to enhance the cellular uptake of siRNA, but high knockdown efficiencies have not been achieved due to endosomal entrapment. In this work, Schiff's base reaction was used to modify a block {P[LAEMA(2-lactobionamidoethyl methacrylamide)37]-b-P[FPMA(4-formyl phenyl methacrylate)2-st-DMA(N,N-dimethylacrylamide)2], P2} and two statistical [P(LAEMA23-st-FPMA3) (P3) and P(LAEMA25-st-FPMA2-st-DMA2) (P4)] aldehyde-based and galactose-based polymers, prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization. An arginine-rich peptide (ARP, KRRKRRRRRK) was used as a cell-penetrating peptide (CPP) and conjugated to the polymers via a Schiff base reaction. The resulting glycopolymer-peptide conjugates were utilized to condense the siRNA to prepare polyplexes with multivalent CPPs (MCPPs, a nanoparticle with multiple copies of the CPP) to enhance the endosomal escape. The polyplexes have different surface properties as determined by the architecture of polymers and the insertion of dimethyl amide moieties. The enhancement of cellular internalization of ARP was observed by labeling the polyplexes with fluorescein isothiocyanate (FITC)-siRNA showing a localization of polyplexes in the cytoplasm of a HeLa (cervical cancer) cell line. In the in vitro EFGR silencing study, the statistical glycopolymer-peptide (P3-P) polyplexes had superior EGFR silencing efficiency in comparison with the other polymers that were studied. Furthermore, P3-P polyplexes led to less off-targeting silencing than lipofectamine 3000. These encouraging results confirmed the potency of decorating galactose-based polymers with CPP, like ARP for their application in siRNA delivery and management of cervical carcinomas.

Temperature-Responsive Aldehyde Hydrogels with Injectable, Self-Healing, and Tunable Mechanical Properties.

Injectable and self-healing hydrogels with exemplary biocompatibility and tunable mechanical properties are urgently needed due to their significant advantages for tissue engineering applications. Here, we report a new temperature-responsive aldehyde hydrogel with dual physical-cross-linked networks and injectable and self-healing properties prepared from an ABA-type triblock copolymer, poly{[FPMA(4-formylphenyl methacrylate)-co-DEGMA[di(ethylene glycol) methyl ether methacrylate]-b-MPC(2-methacryloyloxyethyl phosphorylcholine)-b-(FPMA-co-DEGMA)}. The thermoresponsive poly(DEGMA) segments drive the dehydration and hydrophobic interaction, enabling polymer chain winding as the first cross-linking network, when the temperature is raised above the critical gelation temperature. Meanwhile, the benzaldehyde groups offer physical interactions, including hydrogen bonding and hydrophobic and π-π stacking interactions as the second cross-linking network. When increasing the benzaldehyde content in the triblock copolymers from 0 to 8.2 mol %, the critical gelation temperature of the resulted hydrogels dropped from 35.5 to 19.9 °C and the mechanical modulus increased from 21 to 1411 Pa. Owing to the physical-cross-linked networks, the hydrogel demonstrated excellent injectability and self-healing properties. The cell viabilities tested from MTT assays toward both normal lung fibroblast cells (MRC-5) and cancerous cervical (HeLa) cells were found to be 100 and 101%, respectively, for varying polymer concentrations up to 1 mg/mL. The 3D cell encapsulation of the hydrogels was evaluated by a cytotoxicity Live/Dead assay, showing 92% cell viability. With these attractive physiochemical and biological properties, this temperature-responsive aldehyde hydrogel can be a promising candidate as a cell scaffold for tissue engineering.

Zwitterionic Block Copolymer Prodrug Micelles for pH Responsive Drug Delivery and Hypoxia-Specific Chemotherapy.

Tirapazamine (TPZ) and its derivatives (TPZD) have shown their great potential for efficiently killing hypoxic cancer cells. However, unsatisfactory clinical outcomes resulting from the low bioavailability of the low-molecular TPZ and TPZD limited their further applications. Precise delivery and release of these prodrugs via functional nanocarriers can significantly improve the therapeutic effects due to the targeted drug delivery and enhanced permeability and retention (EPR) effect. Herein, zwitterionic block copolymer (BCP) micelles with aldehyde functional groups are prepared from the self-assembly of poly(2-methacryloyloxyethyl phosphorylcholine-b-poly(di(ethylene glycol) methyl ether methacrylate-co-4-formylphenyl methacrylate) [PMPC-b-P(DEGMA-co-FPMA)]. TPZD is then grafted onto PMPC-b-P(DEGMA-co-FPMA) to obtain a polymer-drug conjugate, PMPC-b-P(DEGMA-co-FPMA-g-TPZD) (BCP-TPZ), through the formation of a pH-responsive imine bond, exhibiting a pH-dependent drug release profile owing to the cleavage of the imine bond under acidic conditions. Outstandingly, BCP-TPZ shows around 13.7-fold higher cytotoxicity to hypoxic cancer cells in comparison to normoxic cancer cells evaluated through an in vitro cytotoxicity assay. The pH-responsiveness and hypoxia-specific cytotoxicity confer BCP-TPZ micelles a great potential to achieve precise delivery of TPZD and thus enhance the therapeutic effect toward tumor-hypoxia.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Facile Preparation of Macromolecular Prodrugs for Hypoxia-Specific Chemotherapy.

Hypoxia-activated prodrugs (HAPs) have emerged as important candidates for chemotherapy due to their efficient killing of hypoxic cancer cells. Traditional small molecule agents, such as tirapazamine (TPZ) and its derivatives, have shown unsatisfactory therapeutic effect in clinical trials due to poor bioavailability in hypoxic tumor regions. Herein, an amphiphilic macromolecular prodrug with hypoxia-specific activity, named as hypoxia-activated macromolecular prodrug (HAMP), is prepared from poly{[poly(ethylene glycol) methacrylate]-st-(methacrylic acid)} [poly(PEGMA-st-MAA)], containing pendant TPZ residues. This polymer can self-assemble in an aqueous system into ∼37 nm sized nanoparticles. In vitro experiments indicated that HAMP shows 5× higher cytotoxicity to hypoxic cancer cells as compared to normoxic cancer cells. Therefore, the developed HAMP can be concurrently used with other therapeutic agents as a highly efficient hypoxia-activated agent.

Oncogenic Epidermal Growth Factor Receptor Silencing in Cervical Carcinoma Mediated by Dynamic Sugar-Benzoxaborole Polyplexes.

Although, various types of pharmaceuticals have been developed for cervical carcinomas, treatment with these drugs often results in a number of undesirable side effects, toxicity and multidrug resistance. Here, we aimed at modifying the genetic profiling of cancer cells by silencing the expression of the epidermal growth factor receptor (EGFR) gene. We have synthesized two kinds of RAFT-made, biocompatible, and cationic polymers for the encapsulation of silencing RNA (siRNA). This vector has a dual capability: it contains a cationic segment to complex with the siRNA and an omega-end modified with an oxaborole group via thiol-ene click chemistry that responds to the acidic tumor microenvironment. This structural innovation enables this macromolecule to interact with multiple polyplexes and release the siRNA in a mild acidic environment. A strategy that has shown enhanced gene silencing without elevating the cytotoxicity of the system, as determined by Western blot analysis. The success of this approach has afforded further interest in utilizing boron-carbohydrate interaction in the development of nonviral vectors for gene therapy.

Hydroxyl-Rich PGMA-Based Cationic Glycopolymers for Intracellular siRNA Delivery: Biocompatibility and Effect of Sugar Decoration Degree.

The ErbB family of proteins, structurally related to the epidermal growth factor receptor (EGFR), is found to be overexpressed in many cancers such as gliomas, a lung and cervical carcinomas. Gene therapy allows to modify the expression of genes like ErbB and has been a promising strategy to target oncogenes and tumor suppressor genes. In the current work, novel hydroxyl-rich poly(glycidyl methacrylate) (PGMA)-based cationic glycopolymers were designed for intracellular small interfering RNA (siRNA) delivery to silence the EGFR gene. The cationic polymers with different sugar decoration degrees (0, 9, and 33%) were synthesized by ring-opening reaction of PGMA with ethanolamine and a lactobionic acid-derived aminosaccharide (Lac-NH2). Specific EGFR knockdown of the protein tyrosine kinase ErbB-overexpressing HeLa cells was achieved using these hydroxyl-rich polycation/siRNA complexes. Higher sugar content improved the biocompatibility of the polymers, but it also seems to decrease the EGFR knockdown capability, which should mainly be related to the surface charge of polyplexes. An optimum balance was observed with PGEL-1 (9% sugar content) formulation, achieving ∼52% knockdown efficiency as well as high cell viability. Considering the specific recognition between galactose residues and asialoglycoprotein receptor in hepatocytes, our novel PGMA-based cationic glycopolymers exhibited promising future to serve as a safe and targeting gene delivery vector to hepatoma cell line like HepG2.

Tumor Microenvironment-Regulated Redox Responsive Cationic Galactose-Based Hyperbranched Polymers for siRNA Delivery.

Tumor microenvironment redox-modulated galactose-based hyperbranched polymers (HRRP) composed of 2-lactobionamidoethylmethacrylamide (LAEMA) and 2-aminoethylmethacrylamide (AEMA) with molecular weights of 10 and 20 kDa and LAEMA:AEMA ratios (L:A) of 1.5 and 1 were prepared via the reversible addition-fragmentation chain transfer (RAFT) polymerization. The remarkable capability of these polymers to respond to the glutathione (GSH) concentration in the tumor environment is the key factor that regulates their cellular internalization and enhances selective siRNA release into the cancer cell cytoplasm. HRRP with a molecular weight of 10 kDa and L:A ratio of 1.5 was capable of forming nanosized polyplexes and achieved around 85% epidermal growth factor receptor (EGFR) silencing in cervical (HeLa) cancer cells in the presence of serum protein without compromising the biocompatibility of the system (around 95% cell viability). The excellent stability of the polyplexes in serum and low cytotoxicity in normal cell lines warrants the use of this redox-responsive galactose-based cationic hyperbranched polymers in gene silencing applications at the preclinical level.

Acid Degradable Cationic Galactose-Based Hyperbranched Polymers as Nanotherapeutic Vehicles for Epidermal Growth Factor Receptor (EGFR) Knockdown in Cervical Carcinoma.

Strong signaling cascades derived from upregulation and overexpression of growth factors such as the EGF-family (epidermal growth factors) have been crucially related to cancer pathogenesis. Gene silencing techniques to modulate the expression of oncogenes and tumor suppresor genes are a strategy that shows great promise for cancer management but still faces some limitations in the design of biocompatible and effective vectors. In this study, we synthesized, by reversible addition-fragmentation chain transfer (RAFT) polymerization, several acid degradable galactose-based hyperbranched cationic polymers with varying molecular weights (10 to 20 kDa) and compositions with 2-lactobioamidoethyl methacrylamide [LAEMA] and 2-aminoethyl methacrylamide hydrochloride [AEMA] at different ratios (2.0, 1.0, and 0.5). These polymers were then evaluated for their ability to enhance Epidermal Growth Factor Receptor (EGFR) knockdown in cervical carcinoma. All the polymer constructs have enhanced capabilities to condensate siRNA (small interfering RNA), showing low toxicity at higher LAEMA:AEMA ratios (1.0 and 2.0). Western blot assays were conducted to quantify the EGFR expression of each treatment group demonstrating superior gene knockdown efficiency for the polymers having a LAEMA:AEMA ratio of 2.0 than the lower ratio counterparts; while maintaining low toxicity levels. Gene silencing of EGFR of up to 60% was achieved with acid degradable polymers having 10 kDa molecular weight and a LAEMA:AEMA ratio of 2.0. The superior stability of the polyplexes under physiological conditions and the low cytotoxicity observed in the 48 h post-transfection demonstrated the high potential of these acid degradable galactose-based hyperbranched cationic polymers for EGFR silencing treatment applications at the clinical level.

Achieving Safe and Highly Efficient Epidermal Growth Factor Receptor Silencing in Cervical Carcinoma by Cationic Degradable Hyperbranched Polymers.

Novel cationic hyperbranched polymers were prepared from 2-aminoethyl methacrylate (AEMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via the reversible addition-fragmentation chain transfer (RAFT) polymerization for siRNA delivery. Non-degradable and acid-degradable hyperbranched polymers were synthesized using N,N'-methylenebis(acrylamide) (MBAm) and 2,2-dimethacroyloxy-1-ethoxypropane (DEP) cross-linkers, respectively. Both types of polymers were capable of forming very stable nanosized polyplexes with siRNA. Epidermal growth factor receptor (EGFR) silencing of 95% was achieved with the acid degradable cationic hyperbranched polymer, and no significant cytotoxicity was observed. Our results confirmed the high potency of using such hyperbranched polymers for the efficient protection and delivery of siRNA.

Effective and Specific Gene Silencing of Epidermal Growth Factor Receptors Mediated by Conjugated Oxaborole and Galactose-Based Polymers.

Oxaborole-based polymers are stimuli-responsive materials that can reversibly interact with diols at pH values higher than their pKa. The strong binding of the oxaborole with cis-hydroxyl groups allow rapid cross-linking of the polymer chains. In this study, we exploited this phenomenon to develop a novel delivery system for the complexation, protection, and delivery of epidermal growth factor receptors (EGFR) siRNA (small interfering RNA). Galactose and oxaborole polymers were first synthesized by the reversible addition-fragmentation chain transfer (RAFT) process, and they were found to show a robust interaction with each other via the oxaborole-diol effect, which allowed the formation of stable polyplexes with siRNA. Although complexes were successfully formed between the neutral galactose and oxaborole-based polymers, these complexes were insufficient in the protection of the siRNA. Therefore, cationic glycopolymers and oxaborole polymers were investigated showing superior complexation with siRNA and exhibiting effective gene silencing in HeLa (cervical) cancer cells, while showing low toxicity. Gene silencing of up to 60% was achieved with these new complexes in the presence and absence of serum. The excellent stability of the complexes under physiological conditions and the observed low cytotoxicity 48 h post-transfection demonstrated the high potential of this new system for gene silencing therapy application in clinics.