Glycopolymers Mediate Suicide Gene Therapy in ASGPR-Expressing Hepatocellular Carcinoma Cells in Tandem with Docetaxel.

Cationic glycopolymers stand out as gene delivery nanosystems due to their inherent biocompatibility and high binding affinity to the asialoglycoprotein receptor (ASGPR), a target receptor overexpressed in hepatocellular carcinoma (HCC) cells. However, their synthesis procedure remains laborious and complex, with problems of solubilization and the need for protection/deprotection steps. Here, a mini-library of well-defined poly(2-aminoethyl methacrylate hydrochloride-co-poly(2-lactobionamidoethyl methacrylate) (PAMA-co-PLAMA) glycopolymers was synthesized by activators regenerated by electron transfer (ARGET) ATRP to develop an efficient gene delivery nanosystem. The glycoplexes generated had suitable physicochemical properties and showed high ASGPR specificity and high transfection efficiency. Moreover, the HSV-TK/GCV suicide gene therapy strategy, mediated by PAMA144-co-PLAMA19-based nanocarriers, resulted in high antitumor activity in 2D and 3D culture models of HCC, which was significantly enhanced by the combination with small amounts of docetaxel. Overall, our results demonstrated the potential of primary-amine polymethacrylate-containing-glycopolymers as HCC-targeted suicide gene delivery nanosystems and highlight the importance of combined strategies for HCC treatment.

Polymer- and lipid-based gene delivery technology for CAR T cell therapy.

Chimeric antigen receptor T cell (CAR T cell) therapy is a revolutionary approach approved by the FDA and EMA to treat B cell malignancies and multiple myeloma. The production of these T cells has been done through viral vectors, which come with safety concerns, high cost and production challenges, and more recently also through electroporation, which can be extremely cytotoxic. In this context, nanosystems can constitute an alternative to overcome the challenges associated with current methods, resulting in a safe and cost-effective platform. However, the barriers associated with T cells transfection show that the design and engineering of novel approaches in this field are highly imperative. Here, we present an overview from CAR constitution to transfection technologies used in T cells, highlighting the lipid- and polymer-based nanoparticles as a potential delivery platform. Specifically, we provide examples, strengths and weaknesses of nanosystem formulations, and advances in nanoparticle design to improve transfection of T cells. This review will guide the researchers in the design and development of novel nanosystems for next-generation CAR T therapeutics.

Novel Non-Viral Vectors Based on Pluronic® F68PEI with Application in Oncology Field.

Copolymers composed of low-molecular-weight polyethylenimine (PEI) and amphiphilic Pluronics® are safe and efficient non-viral vectors for pDNA transfection. A variety of Pluronic® properties provides a base for tailoring transfection efficacy in combination with the unique biological activity of this polymer group. In this study, we describe the preparation of new copolymers based on hydrophilic Pluronic® F68 and PEI (F68PEI). F68PEI polyplexes obtained by doping with free F68 (1:2 and 1:5 w/w) allowed for fine-tuning of physicochemical properties and transfection activity, demonstrating improved in vitro transfection of the human bone osteosarcoma epithelial (U2OS) and oral squamous cell carcinoma (SCC-9) cells when compared to the parent formulation, F68PEI. Although all tested systems condensed pDNA at varying polymer/DNA charge ratios (N/P, 5/1−100/1), the addition of free F68 (1:5 w/w) resulted in the formation of smaller polyplexes (<200 nm). Analysis of polyplex properties by transmission electron microscopy and dynamic light scattering revealed varied polyplex morphology. Transfection potential was also found to be cell-dependent and significantly higher in SCC-9 cells compared to the control bPEI25k cells, as especially evident at higher N/P ratios (>25). The observed selectivity towards transfection of SSC-9 cells might represent a base for further optimization of a cell-specific transfection vehicle.

Engineering silica-polymer hybrid nanosystems for dual drug and gene delivery.

In recent years, it has been shown that a combination of different antitumour strategies involving distinct therapeutic agents, such as chemical compounds and genetic material, could result in an effective therapeutic activity that is much higher than that obtained by conventionally used individual approaches. Therefore, the main goal of this work was to develop a new hybrid nanosystem based on mesoporous silica nanoparticles and polymers to efficiently transport and deliver drug and plasmid DNA into cancer cells. Moreover, its potential to mediate a combinatorial antitumour strategy involving epirubicin and herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy was evaluated. For this purpose, various cationic polymers were assessed, including poly(ß-amino ester) homopolymer, gelatine type A, gelatine type B, and poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate hydrochloride) block copolymer. The obtained results show that using different polymers leads to nanosystems with different physicochemical properties and, consequently, different biological activities. The best formulation was obtained for hybrid nanosystems coated with PEG-b-PAMA. They demonstrated the ability to cotransport and codeliver an anticancer drug and plasmid DNA and effectively mediate the combined antitumour strategy in 2D and 3D tumour cell culture models. In summary, we developed a novel silica- and polymer-based nanosystem able to mediate a dual chemotherapeutic and suicide gene therapy strategy with a much higher therapeutic effect than that obtained through the use of individual approaches, showing its potential for cancer treatment.

Silica-Based Gene Delivery Systems: From Design to Therapeutic Applications.

Advances in gene therapy have been foreshadowing its potential for the treatment of a vast range of diseases involving genetic malfunctioning. However, its therapeutic efficiency and successful outcome are highly dependent on the development of the ideal gene delivery system. On that matter, silica-based vectors have diverted some attention from viral and other types of non-viral vectors due to their increased safety, easily modifiable structure and surface, high stability, and cost-effectiveness. The versatility of silane chemistry and the combination of silica with other materials, such as polymers, lipids, or inorganic particles, has resulted in the development of carriers with great loading capacities, ability to effectively protect and bind genetic material, targeted delivery, and stimuli-responsive release of cargos. Promising results have been obtained both in vitro and in vivo using these nanosystems as multifunctional platforms in different potential therapeutic areas, such as cancer or brain therapies, sometimes combined with imaging functions. Herein, the current advances in silica-based systems designed for gene therapy are reviewed, including their main properties, fabrication methods, surface modifications, and potential therapeutic applications.

Poly(ß-amino ester)-based gene delivery systems: From discovery to therapeutic applications.

Poly(ß-amino ester)s (PßAE) were firstly synthesized in 1983 but only in 2000 these polymers were used for the first time as gene carrier. Thenceforward, due to their excellent gene delivery properties, PßAE were amply explored to afford very effective non-viral vectors. The promising results obtained both in vitro and in vivo studies involving different areas, from cancer therapy to tissue engineering area have aroused a broad interest of the scientific community for this family of biodegradable cationic polymers. This review is the first comprehensive and critical overview of the use of PßAEs as gene carrier. The rational design of PßAEs is a major step aiming to achieve high transfection efficiencies. Moreover, it has been demonstrated that often very small changes in the structure of these polymers have an impressive impact on the transfection efficiency. A critical discussion on the structure performance relationships is presented as well as the outlook for next developments involving these polymers.

Replacing Di(2-ethylhexyl) Terephthalate by Di(2-ethylhexyl) 2,5-Furandicarboxylate for PVC Plasticization: Synthesis, Materials Preparation and Characterization.

The worldwide regulatory demand for the elimination of non-phthalate compounds for poly(vinyl chloride) (PVC) plasticization has intensified the search for alternatives. Concomitantly, sustainability concerns have highlighted sugar-based 2,5-furandicarboxylic acid as one key renewable-chemical for the development of several products, namely di(2-ethylhexyl) 2,5-furandicarboxylate (DEHF) plasticizer. This study addresses the use of DEHF under a realistic scenario of the co-existence of both DEHF and entirely fossil-based plasticizers. More precisely, original PVC blends using mixtures of non-toxic DEHF and di(2-ethylhexyl) terephthalate ester (DEHT) were designed. The detailed structural, thermal, and mechanical characterization of these materials showed that they all have a set of interesting properties that are compatible with those of commercial DEHT, namely a low glass transition (19.2-23.8 °C) and enhanced elongation at break (up to 330%). Importantly, migration tests under different daily situations, such as for example exudation from food/beverages packages and medical blood bags, reveal very low weight loss percentages. For example, in both distilled water and phosphate buffered saline (PBS) solution, weight loss does not exceed ca. 0.3% and 0.2%, respectively. Viability tests show, for the first time, that up to 500 µM of DEHF, a promising cytotoxic profile is observed, as well as for DEHT. Overall, this study demonstrates that the combination of DEHF and DEHT plasticizers result in a noticeable plasticized PVC with an increased green content with promising cytotoxic results.

Poly(ethylene glycol)- block-poly(2-aminoethyl methacrylate hydrochloride)-Based Polyplexes as Serum-Tolerant Nanosystems for Enhanced Gene Delivery.

Incorporation of poly(ethylene glycol) (PEG) into polyplexes has been used as a promising approach to enhance their stability and reduce unwanted interactions with biomolecules. However, this strategy generally has a negative influence on cellular uptake and, consequently, on transfection of target cells. In this work, we explore the effect of PEGylation on biological and physicochemical properties of poly(2-aminoethyl methacrylate) (PAMA)-based polyplexes. For this purpose, different tailor-made PEG- b-PAMA block copolymers, and the respective homopolymers, were synthesized using the controlled/"living" radical polymerization method based on activators regenerated by electron transfer atom transfer radical polymerization. The obtained data show that PEG- b-PAMA-based polyplexes exhibited a much better transfection activity/cytotoxicity relationship than the corresponding non-PEGylated nanocarriers. The best formulation, prepared with the largest block copolymer (PEG45- b-PAMA168) at a 25:1 N/P ratio, presented a 350-fold higher transfection activity in the presence of serum than that obtained with polyplexes generated with the gold standard bPEI. This higher transfection activity was associated to an improved capability to overcome the intracellular barriers, namely the release from the endolysosomal pathway and the vector unpacking and consequent DNA release from the nanosystem inside cells. Moreover, these nanocarriers exhibit suitable physicochemical properties for gene delivery, namely reduced sizes, high DNA protection, and colloidal stability. Overall, these findings demonstrate the high potential of the PEG45- b-PAMA168 block copolymer as a gene delivery system.

Combination of Poly[(2-dimethylamino)ethyl methacrylate] and Poly(ß-amino ester) Results in a Strong and Synergistic Transfection Activity.

This work reports an innovative and very effective gene delivery nanosystem, based on the combination of poly[(2-dimethylamino)ethyl methacrylate] (PDMAEMA) and poly(ß-amino ester) (PßAE) homopolymers, that has the capacity to efficiently deliver genetic material into target cells, even in the presence of serum. The best formulation, prepared with the combination PDMAEMA/4PßAE at the 25/1 nitrogen/phosphate (N/P) ratio, presented a 700-fold and 220-fold higher transfection activity than that obtained with branched polyethylenimine (PEI)-based polyplexes and block copolymer-based polyplexes, respectively. This new nanocarrier revealed high transgene expression in different human cells, including hard-to-transfect normal human astrocytes. The polyplexes presented high protection of genetic material and reduced sizes, which are suitable physicochemical properties for in vivo applications. Overall, this study demonstrates that the combination of PDMAEMA and PßAE homopolymers resulted in a noticeable and synergistic effect in terms of transfection activity, without causing substantial toxicity, constituting a new platform for the development of gene delivery nanosystems.

High transfection efficiency promoted by tailor-made cationic tri-block copolymer-based nanoparticles.

Cationic polymer-based vectors have been considered a promising strategy in gene therapy area due to their inherent ability to condense genetic material and successfully transfect cells. However, they usually exhibit high cytotoxicity. In this work, it is proposed the use of a tailor-made gene carrier based on a tri-block copolymer of poly[2-(dimethylamino)ethyl methacrylate] and poly(ß-amino ester) (PDMAEMA-b-PßAE-b-PDMAEMA), the influence of the PßAE length being assessed. For this purpose, three different block copolymers were prepared varying the molecular weight of this segment. The obtained materials were characterized by NMR and SEC analyzes. Different polyplexes formulations were prepared and evaluated in terms of physicochemical characterization (ethidium bromide intercalation assay, agarose gel electrophoresis assay, dynamic light scattering, zeta potential analyzes and atomic force microscopy) and biological activity (cytotoxicity, and luciferase and green fluorescent protein expression in Hela and COS-7 cell lines). Among the developed nanosystems, the best polyplex formulation revealed between 40- and 60-fold higher transgene expression, in HeLa and COS-7 cell lines, and much lower cytotoxicity than that observed with branched PEI and TurboFect™. Moreover, these nanosystems present suitable physicochemical properties for gene delivery namely reduced mean diameter and high DNA protection. The results reported here show the enormous potential of this block copolymer as gene carrier. STATEMENT OF SIGNIFICANCE: Syntheses of PDMAEMA-b-PßAE-b-PDMAEMA block copolymers for an extremely effective non-viral vector. The block copolymer PDMAEMA3000-b-PßAE12000-b-PDMAEMA3000-based polyplexes at 100/1N/P ratio exhibits between 40- and 60-fold higher transgene expression in HeLa and COS-7 cell lines than commonly used polymeric non-viral vectors, namely branched PEI (known as the gold standard) and TurboFect™ (commercial available).

Novel cationic triblock copolymer of poly[2-(dimethylamino)ethyl methacrylate]-block-poly(ß-amino ester)-block-poly[2-(dimethylamino)ethyl methacrylate]: a promising non-viral gene delivery system.

This manuscript reports the synthesis of a new cationic block copolymer based on poly[2-(dimethylamino)ethyl methacrylate] and poly(ß-amino ester) from different polymerization strategies. For the first time, it is proposed a triblock copolymer based only on cationic segments, aiming a high biocompatibility, enhanced buffering capacity and stimuli-responsive character in a single structure. The new block copolymer successfully condensed the plasmid DNA into nanosized polyplexes. The polyplexes were tested in two different cell lines revealing ∼4-fold and ∼6-fold (in HeLa cells), and ∼11-fold (in COS-7 cells) higher transgene expression than branched PEI and TurboFect™, respectively. These results show that this new block copolymer is a promising candidate to be used as a polymeric non-viral vector.