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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.

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 (25). The observed selectivity towards transfection of SSC-9 cells might represent a base for further optimization of a cell-specific transfection vehicle.

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.

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.

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(β-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.

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 vitro cytotoxicity 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.

Dina Farinha,1,2 Michael Migawa,3 Ana Sarmento-Ribeiro,1,4,5 Henrique Faneca1,2 1CNC - Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal; 2Institute for Interdisciplinary Research (IIIUC), University of Coimbra, Coimbra, Portugal; 3Ionis Pharmaceuticals Inc, Carlsbad, USA; 4Laboratory of Oncobiology and Hematology (LOH) and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 5Clinical Hematology Department, Centro Hospitalar Universitário de Coimbra (CHUC), Coimbra, PortugalCorrespondence: Henrique FanecaCNC - Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, 3004-504, PortugalTel +351-239-820-190Fax +351-239-853-607Email henrique@cnc.uc.ptBackground: 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.Keywords: hepatocellular carcinoma, hybrid nanosystems, drug delivery, GalNAc, sorafenib, selumetinib

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.

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.

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.

This work reports an innovative and very effective gene delivery nanosystem, based on the combination of poly[(2-dimethylamino)ethyl methacrylate] (PDMAEMA) and poly(β-amino ester) (PβAE) 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/4PβAE 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 PβAE 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.

Poly(β-amino ester)s (PβAE) 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, PβAE 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 PβAEs as gene carrier. The rational design of PβAEs 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.

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.

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.

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.

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.

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:PβAE/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 β‑glucan. β‑Glucans 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 β‑glucan 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.

Mitochondria are organelles which play an important role not only in cellular metabolism but also in controlling pathways related with cell death, ionic and redox regulation. Alterations in mitochondrial metabolism are implicated in a variety of diseases, including cancer. Cellular and mitochondrial metabolism are both altered during the different stages of tumor development. As cancer cells have altered metabolic profiles, these alterations are a valid and promising target for anti-cancer agents. We hereby review several molecules that are in different stages of development and which target mitochondria in cancer cells. However, not all compounds are efficiently delivered into mitochondria, especially due to the difficulty of these agents to cross the membranes that surround the organelle, contributing to a loss of effectiveness and specificity. This led to the development of effective strategies aimed at delivering useful cargo to mitochondria, including the use of delocalized lipophilic cations coupled to useful molecules, or peptides that insert in mitochondrial membranes. Although several of those targeting strategies have still a very limited use against cancer cells, we present here the advantages and disadvantages of each combination.

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.

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.

This manuscript reports the synthesis of a new cationic block copolymer based on poly[2-(dimethylamino)ethyl methacrylate] and poly(β-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.

Pancreatic cancer is an aggressive disease and the fourth most lethal cancer in developed countries. Despite all progress in medicine and in understanding the molecular mechanisms of carcinogenesis, pancreatic cancer still has a poor prognosis, the median survival after diagnosis being around 3 to 6 months and the survival rate of 5 years being less than 4%. For pancreatic ductal adenocarcinoma (PDAC), which represents more than 90% of new pancreatic cancer cases, the prognosis is worse than for the other cancers with a patient mortality of approximately 99%. Therefore, there is a pressing need for developing new and efficient therapeutic strategies for pancreatic cancer. In this regard, microRNAs not only have been seen as potential diagnostic and prognostic molecular markers but also as promising therapeutic agents. In this context, this review provides an examination of the most frequently deregulated microRNAs (miRNAs) in PDAC and their putative molecular targets involved in the signaling pathways of pancreatic carcinogenesis. Additionally, it is presented a summary of gene therapy clinical trials involving miRNAs and it is illustrated the therapeutic potential associated to these small non-coding RNAs, for PDAC treatment. The facts presented here constitute a strong evidence of the remarkable opportunity associated to the application of microRNA-based therapeutic strategies as a novel approach for cancer therapy.

The success of gene therapy depends on the efficient delivery of therapeutic genes into target cells in vitro and in vivo. Non-viral vectors, such as cationic liposome–DNA complexes (lipoplexes), have been used for numerous gene delivery applications, although their efficacy is still limited, particularly when compared to that of viral vectors. In this work, we assessed the efficacy of a new gene delivery system generated by non-covalent association of folate to lipoplexes (FA-associated lipoplexes) in two different cancer cell lines (SCC-VII and TSA cells). Association of FA with liposomes composed of DOTAP and cholesterol, and subsequent complexation with DNA greatly increased transfection efficiency above that obtained with plain lipoplexes in both cell lines. The addition of 40 μg of FA to lipoplexes was optimal for transfection and allowed to overcome the inhibitory effect induced by the presence of serum. Notably, the biological activity of the FA-associated complexes was even significantly improved under these conditions. Transfection activity mediated by FA-associated lipoplexes was compared with that by FA-conjugated lipoplexes, and the results showed that electrostatic association of FA to the lipoplexes led to considerably higher levels of biological activity than that involving covalent coupling of FA. Moreover, FA-associated lipoplexes confer greater DNA protection than FA-conjugated lipoplexes. To our knowledge, this is the first study reporting the characterization and application of FA-associated lipoplexes in gene delivery and showing their greater efficacy than that of FA-conjugated lipoplexes. Overall, the results obtained in the present work constitute a strong indication that the developed FA-associated lipoplexes are promising candidates for in vivo gene delivery.

rBbKI and rBbCI, plant recombinant inhibitors from Bauhinia bauhinioides, and BpuTI from Bauhinia purpurea seeds distinctly and specifically block proteolytic enzymes. The secondary structures of those inhibitors were compared and their interactions with phospholipid vesicles were evaluated by the release of calcein and by intrinsic fluorescence of tryptophan residues. The results show that rBbKI, rBbCI and BpuTI are able to interact with phospholipd vesicles and induce membrane permeabilization in a concentration- and pH-dependent manner. The leakage was rapid and extensive at pH 4.5, but at physiological pH, no calcein release was observed. These results may suggest that upon inflammation or microorganism invasion accompanied by lowering of pH, appropriate conditions may occur for the inhibitors to interact with cell membrane and act on specific proteolytic enzyme.

In this work we report biochemical ex vivo studies with a vanadium compound containing a pyridinone ligand, the bis(1,2-dimethyl-3-hydroxy-4-pyridinonate)oxovanadium (IV), V IV O(dmpp) 2 , which has shown to have promising antidiabetic activity. The experiments were carried out on primary adipocytes of 6–8 week old Wistar rats. Insulin-stimulated glucose uptake studies were performed using a radioactive assay by measuring the (U)– 14 C–glucose taken up by the isolated adipocytes for 30 min. Adipocytes were incubated with and without insulin and in the presence and absence of different concentrations of V IV O(dmpp) 2 (100–500 μM) for 45 min. We observed that in a nontoxic concentration, as demonstrated by the Alamar Blue test, V IV O(dmpp) 2 significantly increases glucose uptake, in the absence of insulin, by 5-folds higher than basal, and it has a significant inhibitory effect of 78% on free fatty acid release in isolated adipocytes from normal rats. We also demonstrated that it promotes the phosphorylation of Akt1, a key protein in the insulin signaling cascade. These results were compared with those obtained with another vanadium compound reported in the literature, with a similar structure, the bis(maltolato)oxovanadium (IV) (BMOV), which is now in clinical trials. Our ex vivo results clearly indicate that V IV O(dmpp) 2 is a good candidate to be a promising drug for the treatment of diabetes and other metabolic disorders.

The behaviour of three vanadium(V) systems, namely the pyridinone (V(V)-dmpp), the salicylaldehyde (V(V)-salDPA) and the pyrimidinone (V(V)-MHCPE) complexes, is studied in aqueous solutions, under aerobic and physiological conditions using (51)V NMR, EPR and UV-Visible (UV-Vis) spectroscopies. The speciations for the V(V)-dmpp and V(V)-salDPA have been previously reported. In this work, the system V(V)-MHCPE is studied by pH-potentiometry and (51)V NMR. The results indicate that, at pH ca. 7, the main species present are (V(V)O(2))L(2) and (V(V)O(2))LH(-1) (L=MHCPE(-)) and hydrolysis products, similar to those observed in aqueous solutions of V(V)-dmpp. The latter species is protonated as the pH decreases, originating (V(V)O(2))L and (V(V)O(2))LH. All the V(V)-species studied are stable in aqueous media with different compositions and at physiological pH, including the cell culture medium. The compounds were screened for their potential cytotoxic activity in two different cell lines. The toxic effects were found to be incubation time and concentration dependent and specific for each compound and type of cells. The HeLa tumor cells seem to be more sensitive to drug effects than the 3T3-L1 fibroblasts. According to the IC(50) values and the results on reversibility to drug effects, the V(V)-species resulting from the V(V)-MHCPE system show higher toxicity in the tumor cells than in non-tumor cells, which may indicate potential antitumor activity.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

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(β-amino ester) (PDMAEMA- b -PβAE- b -PDMAEMA), the influence of the PβAE 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 -PβAE- b -PDMAEMA block copolymers for an extremely effective non-viral vector. The block copolymer PDMAEMA 3000 - b -PβAE 12000 - b -PDMAEMA 3000 -based polyplexes at 100/1 N/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).

The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.

Cationic liposome-DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to in vivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of cationic liposomes in clinical applications will be provided.

Mariana Magalhães,1,2,* Dina Farinha,1,* Maria Conceição Pedroso de Lima,1,2 Henrique Faneca1 1Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal *These authors contributed equally tothis work Abstract: Hepatocellular carcinoma (HCC) is the third most common cause of death related to cancer diseases worldwide. The current treatment options have many limitations and reduced success rates. In this regard, advances in gene therapy have shown promising results in novel therapeutic strategies. However, the success of gene therapy depends on the efficient and specific delivery of genetic material into target cells. In this regard, the main goal of this work was to develop a new lipid-based nanosystem formulation containing the lipid lactosyl-PE for specific and efficient gene delivery into HCC cells. The obtained results showed that incorporation of 15% of lactosyl-PE into liposomes induces a strong potentiation of lipoplex biological activity in HepG2 cells, not only in terms of transgene expression levels but also in terms of percentage of transfected cells. In the presence of galactose,which competes with lactosyl-PEfor the binding to theasialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC. In addition, it was found that the incorporation of lactosyl-PEin the nanosystems promotes an increase in their cell binding and uptake. Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application. Overall, the results obtained in this study suggest that the potentiation of the biological activity induced by the presence of lactosyl-PE is due to its specific binding to the ASGP-R, showing that this novel formulation could constitute anew gene delivery nanosystem for application in therapeutic strategies in HCC. Keywords: targeted gene delivery, hepatocellular carcinoma, lactosyl-PE, cationic liposomes, gene delivery nanosystems

This manuscript reports the synthesis of a new cationic block copolymer based on poly[2-(dimethylamino)ethyl methacrylate] and poly(β-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.

New micro three-dimensional (3D) scaffolds using biobased unsaturated polyesters (UPs) were prepared by microstereo-thermal-lithography (μSTLG). This advanced processing technique offers indubitable advantages over traditional printing methods. The accuracy and roughness of the 3D structures were evaluated by scanning electron microscopy and infinite focus microscopy, revealing a suitable roughness for cell attachment. UPs were synthesized by bulk polycondensation between biobased aliphatic diacids (succinic, adipic and sebacic acid) and two different glycols (propylene glycol and diethylene glycol) using fumaric acid as the source of double bonds. The chemical structures of the new oligomers were confirmed by proton nuclear magnetic resonance spectra, attenuated total reflectance Fourier transform infrared spectroscopy and matrix assisted laser desorption/ionization-time of flight mass spectrometry. The thermal and mechanical properties of the UPs were evaluated to determine the influence of the diacid/glycol ratio and the type of diacid in the polyester's properties. In addition an extensive thermal characterization of the polyesters is reported. The data presented in this work opens the possibility for the use of biobased polyesters in additive manufacturing technologies as a route to prepare biodegradable tailor made scaffolds that have potential applications in a tissue engineering area.

Marta Passadouro,1,2 Maria C Pedroso de Lima,1,2 Henrique Faneca11Center for Neuroscience and Cell Biology, 2Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, PortugalAbstract: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and mortal cancer, characterized by a set of known mutations, invasive features, and aberrant microRNA expression that have been associated with hallmark malignant properties of PDAC. The lack of effective PDAC treatment options prompted us to investigate whether microRNAs would constitute promising therapeutic targets toward the generation of a gene therapy approach with clinical significance for this disease. In this work, we show that the developed human serum albumin–1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol/anti-microRNA oligonucleotides (+/–) (4/1) nanosystem exhibits the ability to efficiently deliver anti-microRNA oligonucleotides targeting the overexpressed microRNAs miR-21, miR-221, miR-222, and miR-10in PDCA cells, promoting an almost complete abolishment of microRNA expression. Silencing of these microRNAs resulted in a significant increase in the levels of their targets. Moreover, the combination of microRNA silencing, namely miR-21, with low amounts of the chemotherapeutic drug sunitinib resulted in a strong and synergistic antitumor effect, showing that this combined strategy could be of great importance for therapeutic application in PDAC. Keywords: pancreatic cancer gene therapy, anti-microRNAs oligonucleotides, delivery nanosystems, albumin-associated lipoplexes

Gene therapy is considered a promising approach for the treatment of hepatocellular carcinoma (HCC). In this regard, the main goal of this work was to develop a specific and efficient gene delivery nanosystem to HCC based on 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol cationic liposomes and asialofetuin (ASF), a specific ligand to the asialoglycoprotein receptor (ASGP-R) that is overexpressed in HCC. Our results show that association of ASF to lipoplexes promotes a substantial increase in their biological activity in HCC cells, not only in vitro, but also in an animal model. The transfection activity obtained with this novel nanosystem (ASF-lipoplexes) was much higher than that observed with a highly efficient commercial formulation. On the other hand, the presence of high concentrations of galactose substantially reduced the cell uptake and biological activity of the ASF-lipoplexes. These results, together with those obtained in the presence of inhibitors of endocytosis, show that the potentiation induced by the association of ASF to lipoplexes is due to its specific interaction with the ASGP-R. The physicochemical properties of the generated nanosystem also reinforce this observation. Overall, our results demonstrate for the first time that the novel ASF-lipoplexes present a noticeable ability to specifically and efficiently deliver genetic material into HCC cells.

Great expectations from the application of gene therapy approaches to human disease have been impaired by the unsatisfactory clinical progress observed. Among others, the use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of this promising therapeutic strategy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. This review focuses on the various parameters that are considered to be crucial to optimize the use of cationic lipid-DNA complexes for gene therapy purposes. Particular emphasis is devoted to the analysis of the different stages involved in the transfection process, from the biophysical aspects underlying the formation of the complexes to the different biological barriers that need to be surpassed for gene expression to occur.

Significant progress has been made in the development of different types of nucleic acids, including plasmid DNA, siRNA and oligonucleotides with the potential to form the basis of new treatment options for genetic and acquired diseases. However, the lack of suitable vectors for efficient delivery of nucleic acids into target cells represents a major hurdle for the successful application of gene therapy.

The incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul-ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population.

International audience; Bimodal magnetic resonance imaging (MRI)/optical probes for bioimaging were obtained by grafting two types of lanthanide metal ions, Gd3+ and Eu3+/Tb3+, on the surface of SiO2 nanoparticles. The resulting systems were endowed with relaxometry and photoluminescent properties, respectively. Grafting a pyridine-based aromatic backbone on to the silica surface enhances the emission quantum yield of the Eu3+-containing nanoparticles fivefold compared to similar systems that bear no aromatic antennae. The emission properties of the mixed Ln3+/Gd3+-based nanoparticles are not influenced by the presence of Gd3+. The relaxometric properties of these samples are slightly better than the properties of commercial [Gd(DTPA)]2 (DTPA = diethylenetriaminepentaacetate). When taken up by RAW 264.7 cells (mouse macrophage cell line), such bimodal probes exhibit both T1-weighted MRI increased contrast and fluorescence tracking.

International audience; Suicide gene therapy is based on the introduction into tumor cells of a viral or a bacterial gene, which allows the conversion of a non-toxic compound into a lethal drug. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the huge potential of this strategy when used in combination with new therapeutic approaches. In this review, we summarize the different suicide gene systems and gene delivery vectors addressed to cancer, with particular emphasis on recently developed systems and associated bystander effects. In addition, we review the different strategies that have been used in combination with suicide gene therapy and provide some insights into the future directions of this approach, particularly towards cancer stem cell eradication.

The lack of suitable vectors for efficient nucleic acid delivery into target cells represents a major hurdle for the successful application of gene therapy. Cationic liposomes exhibit attractive features for gene delivery, but their efficacy is still unsatisfactory, particularly for in vivo applications, which justifies the drive to further improve their performance by developing novel and efficient formulations. In the present study, we generated a new formulation of lipoplexes through electrostatic association of folate (FA) to 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes, prepared at different lipid/DNA charge ratios, and explored their potential to mediate gene delivery. The optimal FA-lipoplex formulation was evaluated for its efficacy to mediate antitumoral activity upon application of HSV-tk suicide gene therapy, both in vitro and in an animal model of oral cancer. Our results demonstrate that FA-EPOPC:Chol/DNA lipoplexes were able to promote a great enhancement of transfection and high in vitro antitumoral activity compared to plain lipoplexes in two different cancer cell lines. Most importantly, a considerable reduction of tumor growth was achieved with the developed FA-lipoplexes as compared to that observed for control FA-lipoplexes or plain lipoplexes. Overall, our study shows that FA-EPOPC:Chol/DNA lipoplexes constitute a promising system for the successful application of suicide gene therapy aiming at treating solid tumors.

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.

International audience; The design and synthesis of a combined MRI-optical probe for bio-imaging are reported. The materials studied join the properties of lanthanide (Ln(3+)) complexes and nanoparticles (NPs), offering an excellent solution for bimodal imaging. The hybrid SiO(2)@APS/DTPA:Gd:Ln (Ln = Eu(3+) or Tb(3+)) (APS: 3-aminopropyltriethoxysilane, DTPA: diethylenetriamine pentaacetic acid) system increases the payload of the active magnetic centre (Gd(3+)) and introduces a Ln(3+) long-life excited state (Eu(3+): 0.35 ± 0.02 ms, Tb(3+): 1.87 ± 0.02 ms), with resistance to photobleaching and sharp emission bands. The Eu(3+) ions reside in a single low-symmetry site. Although the photoluminescence emission is not influenced by the simultaneous presence of Gd(3+) and Eu(3+), a moderate r(1) increase and a larger enhancement of r(2) are observed, particularly at high fields, due to susceptibility effects on r(2). The presence of Tb(3+) instead of Eu(3+) further raises r(1) but decreases r(2). These values are constant over a wide (5-13) pH range, indicating the paramagnetic NPs stability and absence of leaching. The uptake of NPs by living cells is fast and results in an intensity increase in the T(1)-weighted MRI images. The optical properties of the NPs in cellular pellets are also studied, confirming their potential as bimodal imaging agents.

Effects of the addition of a cationic amino acid-based synthetic amphiphile, arginine N-lauroyl amide dihydrochloride (ALA), to a lipid-based transfection formulation have been investigated. It is shown that the inclusion of ALA results in a substantial enhancement of the transfection capability of lipoplexes prepared with liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine and cholesterol, which themselves mediate highly efficient transfection. A possible explanation for the increased biological activity is that ALA adsorbed to the surface of the DNA-lipid complexes is involved in triggering internalization. However, in order to identify possible additional factors underlying the enhanced transfection efficiency, the physical properties of formulations with and without ALA were characterized using cryo-transmission electron microscopy, dynamic light scattering, and an ethidium bromide intercalation assay. ALA seems to have limited influence on the initial internal structure of the complexes and the protection of DNA, but its presence is found to decrease the average effective size of the dispersed particles; this change in size may be important in improving the biological activity. Furthermore, ALA can act to influence the transfection efficiency of the formulation by promoting the release of DNA following internalization in the transfected cells.