Targeted downregulation of MYC mediated by a highly efficient lactobionic acid-based glycoplex to enhance chemosensitivity in human hepatocellular carcinoma cells.

The chemosensitization of tumor cells by gene therapy represents a promising strategy for hepatocellular carcinoma (HCC) treatment. In this regard, HCC-specific and highly efficient gene delivery nanocarriers are urgently needed. For this purpose, novel lactobionic acid-based gene delivery nanosystems were developed to downregulate c-MYC expression and sensitize tumor cells to low concentration of sorafenib (SF). A library of tailor-made cationic glycopolymers, based on poly(2-aminoethyl methacrylate hydrochloride) (PAMA) and poly(2-lactobionamidoethyl methacrylate) (PLAMA) were synthesized by a straightforward activators regenerated by electron transfer atom transfer radical polymerization. The nanocarriers prepared with PAMA114-co-PLAMA20 glycopolymer were the most efficient for gene delivery. These glycoplexes specifically bound to the asialoglycoprotein receptor and were internalized through the clathrin-coated pit endocytic pathway. c-MYC expression was significantly downregulated by MYC short-hairpin RNA (MYC shRNA), resulting in efficient inhibition of tumor cells proliferation and a high levels apoptosis in 2D and 3D HCC-tumor models. Moreover, c-MYC silencing increased the sensitivity of HCC cells to SF (IC50 for MYC shRNA + SF 1.9 µM compared to 6.9 µM for control shRNA + SF). Overall, the data obtained demonstrated the great potential of PAMA114-co-PLAMA20/MYC shRNA nanosystems combined with low doses of SF for the treatment of HCC.

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.

Stem cell membrane-coated abiotic nanomaterials for biomedical applications.

Nanoscale materials have been extensively employed for diagnostic and therapeutic purposes. However, the developed nanosystems still suffer from some limitations, namely the rapid elimination by the immune system, lack of targeting to specific cells, and insufficient biocompatibility. Therefore, novel strategies based upon a biomimetic approach have received attention to improving the pharmacokinetics and safety profile of nanosystems. One promising strategy is the application of a biomimetic coating consisting of cell membranes derived from different cell types onto nanoparticle cores. Stem cells have been investigated to develop targeted nanodevices owing to their excellent intrinsic tissue-specific homing features, protecting them from the immune system to reach the sites of inflammation. This targeting ability is conferred by a surface repertoire of stem cell-associated biomolecules. Such nanoscopical materials offer sustained circulation and boosted drug accumulation at target sites, augmenting therapeutic efficacy and safety. Additionally, the coating of nanoparticles with cell membranes acts as a camouflage mechanism to increase their circulation time. The current review explores the particular features of stem cell membrane coating as multifunctional biomimetic surface functionalization agents to camouflage nanoparticle cores. Biomedical applications of engineered stem cell membrane-coated nanoparticles, challenges in clinical translation, and their future prospects are addressed.

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.

Triantennary GalNAc-Functionalized Multi-Responsive Mesoporous Silica Nanoparticles for Drug Delivery Targeted at Asialoglycoprotein Receptor.

In recent years, mesoporous silica particles have been revealed as promising drug delivery systems combining high drug loading capacity, excellent biocompatibility, and easy and affordable synthetic and post-synthetic procedures. In fact, the straightforward functionalization approaches of these particles allow their conjugation with targeting moieties in order to surpass one of the major challenges in drug administration, the absence of targeting ability of free drugs that reduces their therapeutic efficacy and causes undesired side effects. In this context, the main goal of this work was to develop a new targeted mesoporous silica nanoparticle formulation with the capability to specifically and efficiently deliver an anticancer drug to hepatocellular carcinoma (HCC) cells. To this purpose, and as proof of concept, we developed redox-responsive mesoporous silica nanoparticles functionalized with the targeting ligand triantennary N-acetylgalactosamine (GalNAc) cluster, which has high affinity to asialoglycoprotein receptors overexpressed in HCC cells, and loaded them with epirubicin, an anthracycline drug. The produced nanocarrier exhibits suitable physicochemical properties for drug delivery, high drug loading capacity, high biocompatibility, and targeting ability to HCC cells, revealing its biopharmaceutical potential as a targeted drug carrier for therapeutic applications in liver diseases.

Fabrication of 3D scaffolds based on fully biobased unsaturated polyester resins by microstereo-lithography.

Additive Manufacturing (AM) technologies are an effective route to fabricate tailor made scaffolds for tissue engineering (TE) and regenerative medicine, with microstereo-lithography (µSLA) being one of the most promising techniques to produce high quality 3D structures. Here, we report the crosslinking studies of fully biobased unsaturated polyesters (UPs) with 2-hydroxyethyl methacrylate (HEMA) as the unsaturated monomer (UM), using thermal and µSLA crosslinking processes. The resulting resins were fully characterized in terms of their thermal and mechanical properties. Determination of gel content, water contact angle, topography and morphology analysis by atomic force microscopy and scanning electron microscopy were also performed. The results show that the developed UP resins (UPRs) have promising properties for µSLA.In vitrocytotoxicity assays performed with 3T3-L1 cell lines showed that the untreated scaffolds exhibited a maximum cellular viability around 60%, which was attributed to the acidic nature of the UPRs. The treatment of the UPRs and scaffolds with ethanol (EtOH) improved the cellular viability to 100%. The data presented in this manuscript contribute to improve the performance of biobased UPs in AM.

Combining gene therapy with other therapeutic strategies and imaging agents for cancer theranostics.

Cancer is one of the most prevalent and deadly diseases in the world, to which conventional treatment options, such as chemotherapy and radiotherapy, have been applied to overcome the disease or used in a palliative manner to enhance the quality of life of the patient. However, there is an urgent need to develop new preventive and treatment strategies to overcome the limitations of the commonly used approaches. The field of cancer nanomedicine, and more recently the field of nanotheranostics, where imaging and therapeutic agents are combined in a single platform, provide new opportunities for the treatment and the diagnosis of cancer. This combination could bring us closer to a more personalized and cared-for therapy, in opposition to the conventional and standardized approaches. Gene therapy is a promising strategy for the treatment of cancer that requires a transport system to efficiently deliver the genetic material into the target cells. Hence, the main purpose of this work was to review recent findings and developments regarding theranostic nanosystems that incorporate both gene therapy and imaging agents for cancer treatment.

A Combined Antitumor Strategy Mediated by a New Targeted Nanosystem to Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. Sorafenib, which is the first-line therapy for this disease, is associated with reduced therapeutic efficacy that could potentially be overcome by combination with selumetinib. In this context, the main goal of this work was to develop a new nanosystem, composed of a polymeric core coated by a lipid bilayer containing the targeting ligand GalNAc, to specifically and efficiently co-deliver both drugs into HCC cells, in order to significantly increase their therapeutic efficacy. METHODS: The physicochemical characterization of hybrid nanosystems (HNP) and their components was performed by dynamic light scattering, zeta potential, matrix-assisted laser desorption ionization - time of flight mass spectroscopy, and transmission electron microscopy. Cellular binding, uptake and specificity of HNP were evaluated through flow cytometry and confocal microscopy. The therapeutic activity was evaluated namely through: cell viability by the Alamar Blue assay; cell death by flow cytometry using FITC-Annexin V; caspases activity by luminescence; mitochondrial membrane potential by flow cytometry; and molecular target levels by Western blot. RESULTS: The obtained data show that these hybrid nanosystems present high stability and loading capacity of both drugs, and suitable physicochemical properties, namely in terms of size and surface charge. Moreover, the generated formulation allows to circumvent drug resistance and presents high specificity, promoting great cell death levels in HCC cells, but not in non-tumor cells. This potentiation of the antitumor effect of co-loaded drugs was carried out by an increased programmed cell death, being associated with a strong reduction in the mitochondrial membrane potential, a significant increase in the activity of caspases 3/7 and caspase 9, and much greater number of annexin V-positive cells. CONCLUSION: The developed formulation resulted in a high and synergistic antitumor effect, revealing a translational potential to improve therapeutic approaches against HCC.

Non-Viral Gene Delivery Systems.

The advances in the field of gene therapy have significantly improved the possibility for nucleic acids as highly promising agents for the treatment of both inherited and acquired human diseases [...].

Recent advances in peptide-targeted micelleplexes: Current developments and future perspectives.

The decoding of the human genome revolutionized the understanding of how genetics influence the interplay between health and disease, in a multidisciplinary perspective. Thus, the development of exogenous nucleic acids-based therapies has increased to overcome hereditary or acquired genetic-associated diseases. Gene drug delivery using non-viral systems, for instance micelleplexes, have been recognized as promising options for gene-target therapies. Micelleplexes are core-shell structures, at a nanometric scale, designed using amphiphilic block copolymers. These can self-assemble in an aqueous medium, leading to the formation of a hydrophilic and positively charged corona - that can transport nucleic acids, - and a hydrophobic core - which can transport poor water-soluble drugs. However, the performance of these types of carriers usually is hindered by several in vivo barriers. Fortunately, due to a significant amount of research, strategies to overcome these shortcomings emerged. With a wide range of structural features, good stability against proteolytic degradation, affordable characteristic, easy synthesis, low immunogenicity, among other advantages, peptides have increasingly gained popularity as target ligands for non-viral carriers. Hence, this review addresses the use of peptides with micelleplexes illustrating, through the analysis of in vitro and in vivo studies, the potential and future perspectives of this combination.

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.

Supercritically dried superparamagnetic mesoporous silica nanoparticles for cancer theranostics.

Mesoporous silica nanoparticles with a superparamagnetic iron oxide core were prepared in this work, in order to obtain multifunctional platforms with adequate features for cancer theranostics. Three different core-shell nanocomplexes were obtained: IO-OAm/mSiO2, IO-APTES/mSiO2 and IO/SiO2/mSiO2. In the case of IO-OAm/mSiO2 and IO-APTES/mSiO2, iron oxide (IO) was obtained by thermal decomposition, having in this case a coating of oleylamine (OAm) that was in the second formulation exchanged by (3-aminopropyl)triethoxysilane ligand (APTES). Regarding the IO/SiO2/mSiO2 formulation, iron oxide was synthesized by microemulsion. The mesoporous silica shell (mSiO2) on the IO nanoparticles was obtained by sol-gel and the final materials were dried by supercritical fluids drying. VSM confirmed the superparamagnetic behaviour of the nanoparticles, leading to MS of 4.0, 1.8 and 10.2 emu·g-1, for IO-OAm/mSiO2, IO-APTES/mSiO2 and IO/SiO2/mSiO2, respectively. NMR relaxometry has shown the potential of these nanoparticles to be used as T2 contrast agents, with r2 values as high as 63.93 s-1·mM-1 Fe. The three types of nanoparticles exhibited loading contents of epirubicin of ~3% and drug release percentages of 19% for IO-OAm/mSiO2, 24% for IO-APTES/mSiO2 and 31% for IO/SiO2/mSiO2. The cytotoxicity of drug-loaded and non-loaded most promising nanoparticles was assessed, showing high potential of these platforms for application as anticancer drug carriers.

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.

Polymeric nanoengineered HBsAg DNA vaccine designed in combination with ß­glucan.

Antigen-specific immune responses following DNA vaccination are hard to achieve, owing to the difficulty to mediate efficient gene delivery. This study proposed the use of PDMAEMA:PßAE/DNA polyplexes (Pol) as the vehicle of a pDNA vaccine encoding the hepatitis B surface antigen (HBsAg), with these Pol designed in combination with a soluble (Glu) or a particulate (GPs) form of ß­glucan. ß­Glucans are recognized adjuvants that activate immune cells, a good strategy to improve transfection efficiency and vaccine efficacy. Results showed that Pol produced at a 19:1 polymer:DNA (+/-) charge ratio were positively charged (+41 mV), had a mean size of 180 nm and presented high stability under different storage conditions. These polyplexes resulted in enhanced transfection activity than the positive control, showing even higher luciferase gene expression in the presence of GPs (COS-7 and RAW 264.7 cell lines). Additionally, no alterations in hemolysis and plasma coagulation time of human blood were found in the non-cytotoxic working range. Mice vaccination studies (pCMV-S), resulted in a seroconversion rate of 40%, regardless of the additional ß­glucan adjuvants. This work showed the potential of this nanosystem together with GPs to enhance in vitro transfection capacity and to be further studied as a DNA vaccination platform.

Suicide Gene Therapy for Oral Squamous Cell Carcinoma.

Suicide gene therapy has been tested for the treatment of a variety of cancers, including oral cancer. Among the various suicide gene therapy approaches that have been reported, the Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system is one of the most extensively studied systems, holding great promise in cancer therapy. In this chapter, we describe methods to use the HSV-tk/GCV system to achieve antitumor activity, both in cultured oral cancer cells and in orthotopic and subcutaneous murine models of oral squamous cell carcinoma, using ligand-associated lipoplexes for enhancing therapeutic delivery.

Combination of Anti-miRNAs Oligonucleotides with Low Amounts of Chemotherapeutic Agents for Pancreatic Cancer Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the most predominant type of pancreatic cancer and presents one of the highest mortality rates when compared with other carcinomas. The absence of efficient treatment options for PDAC prompted us to investigate whether microRNA inhibition, combined or not with chemotherapeutic agents, would constitute a promising therapeutic approach for this disease. In this chapter, we describe several methods and procedures that can be used to evaluate the potential of new therapeutic strategies involving oligonucleotides against overexpressed microRNAs, in PDAC, either alone or in combination with low amounts of chemotherapeutic drugs.