We developed a formulation of long double-stranded RNA (dsRNA) using interpolyelectrolyte complexes (IPECs) composed of the biopolymers chitosan and alginate, in order to protect the dsRNA from biotic and abiotic factors. Our primary objectives were to enhance stability of dsRNA against environmental nucleases and, secondarily, to mitigate the negative charge of the dsRNA, which may promote foliar uptake. Our approach relies on submicron particles with adjustable surface charge being either positive or negative. Following this approach, we obtained a high encapsulation efficiency of 94 %. Subsequently, we investigated the influence of the charge ratio and total polymer content on the size, size distribution and ζ-potential of the IPECs. We discovered that formulating at low polymer concentrations ≤0.05 g/L with charge ratios of ≤0.9 (+/-) and ≥ 1.25 (+/-), respectively, produced <100 nm particles. Furthermore, the IPEC formulation protected dsRNA from enzymatic degradation by RNase III and micrococcal nuclease. In addition, we observed outstanding protection of formulated dsRNA from heat degradation. Experiments on Nicotiana benthamiana plants showed that formulated dsRNA offered protection against tobacco mosaic virus. In essence, this formulation demonstrates versatility for the production of IPECs with customizable size, surface charge, and nucleic acid content.

Historically, RNA delivery via nanoparticles has primarily relied on encapsulation, as demonstrated by lipid nanoparticles in SARS-CoV-2 vaccines. Concerns about RNA degradation on nanoparticle surfaces initially limited the exploration of adsorption-based approaches. However, recent advancements have renewed interest in adsorption as a viable alternative. This Viewpoint explores the approaches of RNA incorporation in nanoparticles, comparing encapsulation, adsorption, and the combination of encapsulation and adsorption, and presents a framework to guide the selection of the most suitable strategy based on general characteristics.

Ovarian cancer, characterized by high lethality, presents a significant clinical challenge. The standard first-line treatment is surgery and chemotherapy; however, postoperative chemotherapy is often ineffective and associated with severe side effects and the development of drug resistance. Consequently, there is an urgent need for innovative drug delivery strategies to enhance therapeutic efficacy. Natural polysaccharide polymers with high bioactivity have been extensively investigated for use alone or as adjuvants to chemotherapy and radiotherapy, and also for the preparation of efficient delivery systems for ovarian cancer therapy. This paper aims to review recent advances in the application of natural polysaccharides, including hyaluronic acid, chitosan, alginate, and cellulose, in the therapy of ovarian cancer. This paper primarily discusses the anti-tumor properties inherent to these natural polysaccharide polymers and offers a summary of their role in delivery systems used in ovarian cancer therapy.

Here, a pulmonary formulation based on lipid-polymer hybrid nanoparticles carrying small interfering RNA (siRNA) was developed to realize a RNA interference-based therapy to treat respiratory diseases. Toward this aim, a new copolymer was synthesized, by functionalization of the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide with 35 mol % of 1,2-bis(3-aminopropylamino)ethane, 0.4 mol % of fluorescent dye, and 4.5 mol % of poly(lactic-co-glycolic acid). This was used to encapsulate siRNA targeting the green fluorescent protein (siGFP), within a lipid shell made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-(polyethylene glycol)2000. siGFP-loaded lipid-polymer hybrid nanoparticles (LPHFNPs@siGFP) exhibited colloidal size (∼164 nm), positive ζ potential, high siRNA encapsulation efficiency (∼99%), and a core-shell morphology. They showed high cellular uptake and a gene silencing efficiency of ∼50% in human lung cancer cells expressing GFP. To address aerodynamic challenges, LPHFNPs@siGFP were spray-dried with trehalose, yielding spherical particles (∼3 μm) with 80% siRNA encapsulation efficiency, excellent aerosolization properties, and a gene silencing efficiency comparable to the fresh LPHFNPs@siGFP sample.

The gene therapy has been developed into a new cancer treatment option. Now that we know which molecular components contribute to carcinogenesis, we may use gene therapy to target particular signalling pathways in cancer treatment. Problems with gene therapy include genetic tool degradation in blood, off-targeting features, and inadequate tumor site accumulation; new delivery mechanisms are needed to address these issues. A polysaccharide made from chitin, chitosan has found extensive use in the creation of nanoparticles. The delivery of genes in the treatment of illnesses, particularly cancer, has made use of nanostructures modified with chitosan. Topics covered in this review center on cancer treatment using chitosan-based polymers for siRNA delivery. This study aims to assess the potential of chitosan nanoparticles for the simultaneous administration of siRNA and anti-cancer medications. In cancer treatment, these nanoparticles can transport phytochemicals or chemotherapeutics together with siRNA. In addition, chitosan nanoparticles loaded with siRNA can inhibit the growth and spread of human malignancies by delivering siRNA that targets particular genes. Chitosan nanoparticles loaded with siRNA can heighten the responsiveness of cancer cells. Future therapeutic applications of chitosan nanoparticles may open the path for cancer treatment, thanks to their biocompatibility and biosafety.

Resolving the sequence and structure of flexible biomolecules such as DNA is crucial to understanding their biological mechanisms and functions. Traditional structural biology methods remain challenging for the analysis of small and disordered biomolecules, especially those that are difficult to label or crystallize. Recent development of single-molecule tip-enhanced Raman spectroscopy (TERS) offers a label-free approach to identifying nucleobases in a single DNA chain. However, a clear demonstration of sequencing both spatially and spectrally at single-base resolution is still elusive due to the challenges caused by weak Raman signals and the flexibility of DNA molecules. Here, we report a proof-of-principle demonstration to this end, spectrally resolving in real space individual nucleobases and their sequence structures within a short, single-stranded DNA molecule artificially designed. This breakthrough is achieved through the development of subnanometer-resolved low-temperature TERS methodology for such thermally unstable flexible biomolecules. Further TERS mapping over individual nucleobases provides additional structural information about the molecular configurations and even the locations of functional groups, offering a way to track modification types and binding sites in biomolecules.

Over the past years, gene therapeutics have held great promise for treating many inherited and acquired diseases. The increasing number of approved gene therapeutics and developing clinical pipelines demonstrate the potential to treat diseases by modifying their genetic blueprints in vivo. Compared with conventional treatments targeting proteins rather than underlying causes, gene therapeutics can achieve enduring or curative effects via gene activation, inhibition, and editing. However, the delivery of DNA/RNA to the target cell to alter the gene expression is a complex process that involves, crossing numerous barriers in both the extracellular and intracellular environment. Generally, the delivery strategies can be divided into viral-based and non-viral-based vectors. This review summarizes various bioanalysis strategies that support the non-virus-based gene therapeutics research, including pharmacokinetics (PK)/toxicokinetics (TK), biodistribution, immunogenicity evaluations for the gene cargo, vector, and possible expressed protein, and highlights the challenges and future perspectives of bioanalysis strategies in non-virus-based gene therapeutics. This review may provide new insights and directions for the development of emerging bioanalytical methods, offering technical support and a research foundation for innovative gene therapy treatments.

Chemical-intensive agriculture challenges environmental sustainability and biodiversity and must be changed. Minimizing the use of agrochemicals based on renewable resources can reduce or eliminate ecosystems and biodiversity threats. Nanochitosan as a sustainable alternative offers promising solutions for sustainable agricultural practices that work at multiple spatial and temporal scales throughout the plant growth cycle. This review focuses on the potential of nanochitosan in sustainable agricultural production and provides insights into the mechanisms of action and application options of nanochitosan throughout the plant growth cycle. We emphasize the role of nanochitosan in increasing crop yields, mitigating plant diseases, and reducing agrochemical accumulation. The paper discusses the sources of nanochitosan and its plant growth promotion, antimicrobial properties, and delivery capacity. Furthermore, we outline the challenges and prospects of research trends of nanochitosan in sustainable agricultural production practices and highlight the potential of nanochitosan as a sustainable alternative to traditional agrochemicals.

Nanotechnology has transformed the oncology sector by particularly targeting cancer cells and enhancing the efficacy of conventional therapies, not only improving efficacy of conventional therapeutics, but also reducing systemic toxicity. Environmentally friendly materials are the top choice for treating cancer. Chitosan, sourced from chitin, is widely used with its derivatives for the extensive synthesis or modification of nanostructures. Chitosan has been deployed to develop hydrogels, as 3D polymeric networks capable of water absorption with wide biomedical application. The chitosan hydrogels are biocompatible and biodegradable structures that can deliver drugs, genes or a combination of them in cancer therapy. Increased tumor ablation, reducing off-targeting feature and protection of genes against degradation are benefits of using chitosan hydrogels in cancer therapy. The efficacy of cancer immunotherapy can be improved by chitosan hydrogels to prevent emergence of immune evasion. In addition, chitosan hydrogels facilitate photothermal and photodynamic therapy for tumor suppression. Chitosan hydrogels can synergistically integrate chemotherapy, immunotherapy, and phototherapy in cancer treatment. Additionally, chitosan hydrogels that respond to stimuli, specifically thermo-sensitive hydrogels, have been developed for inhibiting tumors.

Recent cancer therapy research has found that chitosan (Ch)-based nanoparticles show great potential for targeted gene delivery. Chitosan, a biocompatible and biodegradable polymer, has exceptional properties, making it an ideal carrier for therapeutic genes. These nanoparticles can respond to specific stimuli like pH, temperature, and enzymes, enabling precise delivery and regulated release of genes. In cancer therapy, these nanoparticles have proven effective in delivering genes to tumor cells, slowing tumor growth. Adjusting the nanoparticle's surface, encapsulating protective agents, and using targeting ligands have also improved gene delivery efficiency. Smart nanoparticles based on chitosan have shown promise in improving outcomes by selectively releasing genes in response to tumor conditions, enhancing targeted delivery, and reducing off-target effects. Additionally, targeting ligands on the nanoparticles' surface increases uptake and effectiveness. Although further investigation is needed to optimize the structure and composition of these nanoparticles and assess their long-term safety, these advancements pave the way for innovative gene-focused cancer therapies.

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule's chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions.

Lung cancer is the leading cause of cancer-related mortality worldwide, largely due to the limited efficacy of anticancer drugs, which is primarily attributed to insufficient doses reaching the lungs. Additionally, patients undergoing treatment experience severe systemic adverse effects due to the distribution of anticancer drugs to non-targeted sites. In light of these challenges, there has been a growing interest in pulmonary administration of drugs for the treatment of lung cancer. This route allows drugs to be delivered directly to the lungs, resulting in high local concentrations that can enhance antitumor efficacy while mitigating systemic toxic effects. However, pulmonary administration poses the challenge of overcoming the mechanical, chemical, and immunological defenses of the respiratory tract that prevent the inhaled drug from properly penetrating the lungs. To overcome these drawbacks, the use of nanoparticles in inhaler formulations may be a promising strategy. Nanoparticles can assist in minimizing drug clearance, increasing penetration into the lung epithelium, and enhancing cellular uptake. They can also facilitate increased drug stability, promote controlled drug release, and delivery to target sites, such as the tumor environment. Among them, chitosan-based nanoparticles demonstrate advantages over other polymeric nanocarriers due to their unique biological properties, including antitumor activity and mucoadhesive capacity. These properties have the potential to enhance the efficacy of the drug when administered via the pulmonary route. In view of the above, this paper provides an overview of the research conducted on the delivery of anticancer drug-loaded chitosan-based nanoparticles incorporated into inhaled drug delivery devices for the treatment of lung cancer. Furthermore, the article addresses the use of emerging technologies, such as siRNA (small interfering RNA), in the context of lung cancer therapy. Particularly, recent studies employing chitosan-based nanoparticles for siRNA delivery via the pulmonary route are described.

Gene therapy uses modern molecular biology methods to repair disease-causing genes. As a burgeoning therapeutic, it has been widely applied for cancer therapy. Since 1989, there have been numerous clinical gene therapy cases worldwide. However, a few are successful. The main challenge of clinical gene therapy is the lack of efficient and safe vectors. Although viral vectors show high transfection efficiency, their application is still limited by immune rejection and packaging capacity. Therefore, the development of non-viral vectors is overwhelming. Nanoplatform-based non-viral vectors become a hotspot in gene therapy. The reasons are mainly as follows. 1) Non-viral vectors can be engineered to be uptaken by specific types of cells or tissues, providing effective targeting capability. 2) Non-viral vectors can protect goods that need to be delivered from degradation. 3) Nanoparticles can transport large-sized cargo such as CRISPR/Cas9 plasmids and nucleoprotein complexes. 4) Nanoparticles are highly biosafe, and they are not mutagenic in themselves compared to viral vectors. 5) Nanoparticles are easy to scale preparation, which is conducive to clinical conversion and application. Here, an overview of the categories of nanoplatform-based non-viral gene vectors, the limitations on their development, and their applications in cancer therapy.

Gene therapy involves the introduction of exogenous genetic material into host tissues to modify gene expression or cellular properties for therapeutic purposes. Initially developed to address genetic disorders, gene therapy has expanded to encompass a wide range of conditions, notably cancer. Effective delivery of nucleic acids into target cells relies on carriers, with non-viral systems gaining prominence due to their enhanced safety profile compared to viral vectors. Chitosan, a biopolymer, is frequently utilized to fabricate nanoparticles for various biomedical applications, particularly nucleic acid delivery, with recent emphasis on targeting cancer cells. Chitosan's positively charged amino groups enable the formation of stable nanocomplexes with nucleic acids and facilitate interaction with cell membranes, thereby promoting cellular uptake. Despite these advantages, chitosan-based nanoparticles face challenges such as poor solubility at physiological pH, non-specificity for cancer cells, and inefficient endosomal escape, limiting their transfection efficiency. To address these limitations, researchers have focused on enhancing the functionality of chitosan nanoparticles. Strategies include improving stability, enhancing targeting specificity, increasing cellular uptake efficiency, and promoting endosomal escape. This review critically evaluates recent formulation approaches within these categories, aiming to provide insights into advancing chitosan-based gene delivery systems for improved efficacy, particularly in cancer therapy.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Cartilage repair has been a significant challenge in orthopedics that has not yet been fully resolved. Due to the absence of blood vessels and the almost cell-free nature of mature cartilage tissue, the limited ability to repair cartilage has resulted in significant socioeconomic pressures. Polysaccharide materials have recently been widely used for cartilage tissue repair due to their excellent cell loading, biocompatibility, and chemical modifiability. They also provide a suitable microenvironment for cartilage repair and regeneration. In this Review, we summarize the techniques used clinically for cartilage repair, focusing on polysaccharides, polysaccharides for cartilage repair, and the differences between these and other materials. In addition, we summarize the techniques of tissue engineering strategies for cartilage repair and provide an outlook on developing next-generation cartilage repair and regeneration materials from polysaccharides. This Review will provide theoretical guidance for developing polysaccharide-based cartilage repair and regeneration materials with clinical applications for cartilage tissue repair and regeneration.

The ocean is the common home of a large number of marine organisms, including plants, animals, and microorganisms. Researchers can extract thousands of important bioactive components from the oceans and use them extensively to treat and prevent diseases. In contrast, marine polysaccharide macromolecules such as alginate, carrageenan, Laminarin, fucoidan, chitosan, and hyaluronic acid have excellent physicochemical properties, good biocompatibility, and high bioactivity, which ensures their wide applications and strong therapeutic potentials in drug delivery. Drug delivery systems (DDS) based on marine polysaccharides and modified marine polysaccharide molecules have emerged as an innovative technology for controlling drug distribution on temporal, spatial, and dosage scales. They can detect and respond to external stimuli such as pH, temperature, and electric fields. These properties have led to their wide application in the design of novel drug delivery systems such as hydrogels, polymeric micelles, liposomes, microneedles, microspheres, etc. In addition, marine polysaccharide-based DDS not only have smart response properties but also can combine with the unique biological properties of the marine polysaccharide base to exert synergistic therapeutic effects. The biological activities of marine polysaccharides and the design of marine polysaccharide-based DDS are reviewed. Marine polysaccharide-based responsive DDS are expected to provide new strategies and solutions for disease treatment.

In this study, a simple novel hybrid mesoporous nanomaterial derived from a metal-organic framework (ZIF-8) and chitosan, which were coated on green bismuth oxide, has been successfully synthesized, characterized, and applied to investigate its dapsone loading-releasing capability in the aqueous media. This suggested nanocomposite showed promise for drug loading from water b using hydrogen bonds, pi-pi, and electrostatic interactions. Structural and morphological analyses were performed on the proposed green synthesized nanocomposite through scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, Brunauer-Emmett-Teller analysis, and thermogravimetric analysis. Various influencing parameters, including pH, nanocomposite dose, and contact time, were investigated to optimize the dapsone loading process. Utilizing the non-linear optimization methodology, the results show that dapsone-loading efficiency was >85 % for 50 mg.L-1 of dapsone drug. The optimum parameters for achieving maximal loading of dapsone drug were pH = 6.8, hybrid mesosphere dose = 2.6 mg.mL-1, and time = 53 min. Based on the release investigations, the dapsone-loaded nanocomposite was put into phosphate buffer saline, at pH = 7.4 and T = 37 °C, with a maximum efficiency of 93.9 after 24 h.

Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery.

One of the critical steps in gene therapy is the successful delivery of the genes. Immunogenicity and toxicity are major issues for viral gene delivery systems. Thus, non-viral vectors are explored. A cationic polysaccharide like chitosan could be used as a nonviral gene delivery vector owing to its significant interaction with negatively charged nucleic acid and biomembrane, providing effective cellular uptake. However, the native chitosan has issues of targetability, unpacking ability, and solubility along with poor buffer capability, hence requiring modifications for effective use in gene delivery. Modified chitosan has shown that the "proton sponge effect" involved in buffering the endosomal pH results in osmotic swelling owing to the accumulation of a greater amount of proton and chloride along with water. The major challenges include limited exploration of chitosan as a gene carrier, the availability of high-purity chitosan for toxicity reduction, and its immunogenicity. The genetic drugs are in their infancy phase and require further exploration for effective delivery of nucleic acid molecules as FDA-approved marketed formulations soon.

Cancer development and progression of cancer are closely associated with the activation of oncogenes and loss of tumor suppressor genes. Nucleic acid drugs (e.g., siRNA, mRNA, and DNA) are widely used for cancer therapy due to their specific ability to regulate the expression of any cancer-associated genes. However, nucleic acid drugs are negatively charged biomacromolecules that are susceptible to serum nucleases and cannot cross cell membrane. Therefore, specific delivery tools are required to facilitate the intracellular delivery of nucleic acid drugs. In the past few decades, a variety of nanoparticles (NPs) are designed and developed for nucleic acid delivery and cancer therapy. In particular, the polymeric NPs in response to the abnormal redox status in cancer cells have garnered much more attention as their potential in redox-triggered nanostructure dissociation and rapid intracellular release of nucleic acid drugs. In this review, the important genes or signaling pathways regulating the abnormal redox status in cancer cells are briefly introduced and the recent development of redox-responsive NPs for nucleic acid delivery and cancer therapy is systemically summarized. The future development of NPs-mediated nucleic acid delivery and their challenges in clinical translation are also discussed.

MicroRNAs (miRNAs) have a crucial role in the regulation of gene expression in tumor development, invasion, and metastasis. Herein, miRNA-340 (miR-340) has been shown to play tumor suppressor activity in breast cancer (BC). However, the clinical applications of miRNAs request the development of safe and effective delivery systems capable of protecting nucleic acids from degradation. In this study, biodegradable chitosan nanoparticles incorporating miR-340 plasmid DNA (pDNA) (miR-340 CNPs) were synthesized and characterized. Then, the anti-tumor effects of miR-340 CNPs were investigated using 4T1 BCE cells. The spherical nanoparticles (NPs) with an appropriate mean diameter of around 266 ± 9.3 nm and zeta potential of +17 ± 1.8 mV were successfully prepared. The NPs showed good stability, high entrapment efficiency and a reasonable release behavior, meanwhile their high resistance against enzymatic degradation was verified. Furthermore, NPs demonstrated appropriate transfection efficiency and could induce apoptosis, so had toxicity in 4T1 BCE cells. Also, CD47 expression on the surface of cancer cells was significantly reduced after treatment with miR-340 CNPs. The results showed that miR-340 CNPs augmented the expression of P-27 in BC cells. Furthermore, miR-340 CNPs caused down-regulation of BRP-39 (breast regression protein-39) increasingly suggested as a prognostic biomarker for neoplastic diseases like BC. In conclusion, our data show that miR-340 CNPs can be considered as a promising new platform for BC gene therapy.

Cancer-cell-specific fluorescent photosensitizers (PSs) are highly desired molecular tools for cancer ablation with minimal damage to normal cells. However, such PSs that can achieve cancer specification and ablation and a self-reporting manner concurrently are rarely reported and still an extremely challenging task. Herein, we have proposed a feasible strategy and conceived a series of fluorescent PSs based on simple chemical structures for identifying and killing cancer cells as well as monitoring the photodynamic therapy (PDT) process by visualizing the change of subcellular localization. All of the constructed cationic molecules could stain mitochondria in cancer cells, identify cancer cells specifically, and monitor cancer cell viability. Among these, IVP-Br has the strongest ability to produce ROS, which serves as a potent PS for specific recognition and killing of cancer cells. IVP-Br could translocate from mitochondria to the nucleolus during PDT, self-reporting the entire therapeutic process. Mechanism study confirms that IVP-Br with light irradiation causes cancer cell ablation via inducing cell cycle arrest, cell apoptosis, and autophagy. The efficient ablation of tumor through PDT induced by IVP-Br has been confirmed in the 3D tumor spheroid chip. Particularly, IVP-Br could discriminate cancer cells from white blood cells (WBCs), exhibiting great potential to identify circulating tumor cells (CTCs).

RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA molecules to inhibit the expression of disease-causing genes specifically. In other words, it silences genes and suppresses the transcription process. The main theory behind RNA therapy is that RNA molecules can prevent the translation into proteins by binding to specific messenger RNA (mRNA) molecules. By targeting disease-related mRNA molecules, RNA therapy can effectively silence or reduce the development of harmful proteins. There are different types of RNA molecules used in therapy, including small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamer, ribozyme, and antisense oligonucleotides (ASOs). These molecules are designed to complement specific mRNA sequences, allowing them to bind and degrade the targeted mRNA or prevent its translation into protein. Nanotechnology is also highlighted to increase the efficacy of RNA-based drugs. In this chapter, while examining various methods of RNA therapy, we discuss the advantages and challenges of each.

Cytotoxicity, speedy degradation, and limited cellular absorption are the foremost features influencing the successful delivery of RNAs. Chitosan (Cs) is a polymer that offers an advantage due to its bio-compatibility and biodegradable nature, making it an ideal polycationic vector for delivering siRNA. In this study, chitosan has been modified with arginine in order to increase its encapsulation of siRNA and improve cellular absorption. It was discovered that arginine and guanidino moieties could transport through membranes of cells and play an important part in membrane permeability. FTIR and 13C NMR were used to characterize the complex. These chitosan-arginine (CsAr) siRNA complexes are further encapsulated in anionic DPPC/cholesterol liposomes to combine the effects of liposome-chitosan-arginine complexes called lipopolyplexes (LCAr). Formed LCAr were investigated for their lipid/CsAr-siRNA ratios, size, zeta-potential, heparin, and serum RNase stability by agarose gel retardation, and cell uptake efficiency compared to their "parent" polyplexes. Results revealed complete lipidation of CsAr-siRNA polyplexes at lipid mass ratio 10 resulting in lipopolyplexes in the 120 to 230nm range. Polyplex entrapped ~70% of siRNA, whereas lipidation increases siRNA encapsulation to ~95%. Developed LCAr showed ~4 times less hemolytic potential as compared to the parent polyplexes at the highest siRNA dose. The CsAr-siRNA and its lipid-coated form showed enhanced cellular association as compared to the marketed Lipofectamine 2000 proving its effectiveness in siRNA delivery. CsAr-liposome conjugation is simple and safe, and serves as a robust carrier for gene transport in physiological situations without compromising transfection efficacy.

Spinal cord injury (SCI) is a central system disease with a high rate of disability. Pathological changes such as ischemia and hypoxia of local tissues, oxidative stress and apoptosis could lead to limb pain, paralysis and even life-threatening. It was reported that catalase (CAT) was the main antioxidant in organisms, which could remove reactive oxygen species (ROS) and release oxygen (O2). However, the efficacy of the drug is largely limited due to its poor stability, low bioavailability and inability to cross the blood spinal cord barrier (BSCB). Therefore, in this study, we prepared folic acid-functionalized chitosan nanoparticles to deliver CAT (FA-CSNCAT) for solving this problem. In vivo small animal imaging results showed that FA-CSN could carry CAT across the BSCB and target to the inflammatory site. In addition, Immunofluorescence, ROS assay and JC-1 probe were used to detect the therapeutic effect of FA-CSNCAT in vitro and in vivo. The results showed that FA-CSNCAT could alleviate the hypoxic environment at the injured site and remove ROS, thereby inhibiting oxidative stress and protecting neurons, which may provide a new idea for clinical medication of SCI.

Gene delivery via the oral route offers a promising strategy for improving DNA vaccination and gene-based therapy outcomes. The noninvasive nature of oral delivery lends to ease of dosing, which can facilitate convenience and patient compliance. Moreover, oral administration allows for both local and systemic production of therapeutic genes or, in the case of DNA vaccination, mucosal and systemic immunity. Here, we describe the methods to produce a dual biomaterial, oral DNA delivery system composed of chitosan (CS) and zein (ZN). In this system, CS serves to encapsulate and deliver DNA cargo to intestinal cells in the form of CS-DNA nanoparticles (CS-DNA NPs), while ZN is used to form a protective matrix around the CS-DNA NPs that prevent degradation during gastric transit but then degrades to release the CS-DNA NPs for transfection upon entry into the intestines. These particles have demonstrated the ability to effectively protect cargo DNA from simulated gastric degradation in vitro and mediate transgene production in vivo, making them an effective oral gene delivery system.

Intranasal (i.n.) vaccination with adjuvant-free plasmid DNA encoding the leishmanial antigen LACK (LACK DNA) has shown to induce protective immunity against both cutaneous and visceral leishmaniasis in rodents. In the present work, we sought to evaluate the safety and effectiveness of d,l-glyceraldehyde cross-linked chitosan microparticles (CCM) as a LACK DNA non-intumescent mucoadhesive delivery system. CCM with 5 μm of diameter was prepared and adsorbed with a maximum of 2.4 % (w/w) of DNA with no volume alteration. Histological analysis of mouse nostrils instilled with LACK DNA / CCM showed microparticles to be not only mucoadherent but also mucopenetrant, inducing no local inflammation. Systemic safeness was confirmed by the observation that two nasal instillations one week apart did not alter the numbers of bronchoalveolar cells or blood eosinophils; did not alter ALT, AST and creatinine serum levels; and did not induce cutaneous hypersensitivity. When challenged in the footpad with Leishmania amazonensis, mice developed significantly lower parasite loads as compared with animals given naked LACK DNA or CCM alone. That was accompanied by increased stimulation of Th1-biased responses, as seen by the higher T-bet / GATA-3 ratio and IFN-γ levels. Together, these results demonstrate that CCM is a safe and effective mucopenetrating carrier that can increase the efficacy of i.n. LACK DNA vaccination against cutaneous leishmaniasis.

This literature review aims to update the current knowledge on toxicity of chitosan nanoparticles, compare the recent findings and identify the gaps with knowledge that is present for the chitosan nanoparticles.

The publications between 2010 and 2020 were searched in Science Direct, Pubmed.gov, Google Scholar, Research Gate, and ClinicalTrials.gov, according to the inclusion and exclusion criteria. 30 primary research studies were obtained from the literature review to compare the in vitro in vivo toxicity profiles among the chitosan nanoparticles.

Chitosan nanoparticles and other types of nanoparticles show cytotoxic effects on cancer cells while having minimal toxicity on normal cells. This apparent effect poses some considerations for use in incorporating cancer therapeutics into chitosan nanoparticles as an administration form. The concentration, duration of exposure, and pH of the solution can influence nanoparticle cytotoxicity, particularly in zebrafish. Different cell lines exhibit varying degrees of toxicity when exposed to nanoparticles, and of note are liver cells that show toxicity under exposure as indicated by increased alanine transaminase (ALT) levels. Aside from ALT, platelet aggregation can be considered a toxicity induced by chitosan nanoparticles. In addition, zebrafish cells experience the most toxicity, including organ damage, neurobehavioral impairment, and developmental abnormalities, when exposed to nanoparticles. However, nanoparticles may exhibit different toxicity profiles in different organisms, with brain toxicity and liver toxicity being present in zebrafish but not rats. Different organs exhibit varying degrees of toxicity, with the eye and mouth apparently having the lowest toxicity, while the brain, intestine, muscles and lung showing mixed results. Cardiotoxicity induced by chitosan nanoparticles was not observed in zebrafish embryos, and nanoparticles may reduce cardiotoxicity when delivering drug. Toxicity found in an organ may not necessarily mean that it is toxic towards all the cells found in that organ, as muscle toxicity was present when tested in zebrafish but not in C2C12 myoblast cells. Some of the studies conducted may have limitations that need to be reconsidered to account for differing results, with some examples being two experiments done on HeLa cells where one study concluded chitosan nanoparticles were toxic to the cells while the other seems to have no toxicity present. With regards to LD50, one study has stated the concentration of 64.21 mg/ml was found. Finally, smaller nanoparticles generally exhibit higher toxicity in cells compared to larger nanoparticles.

This literature review did not uncover any published clinical trials with available results. Subsequent research endeavors should prioritize conducting clinical trials involving human volunteers to directly assess toxicity, rather than relying on cell or animal models.

Nanocarriers are widely used for efficient delivery of different cargo into mammalian cells; however, delivery into plant cells remains a challenging issue due to physical and mechanical barriers such as the cuticle and cell wall. Here, we discuss recent progress on biodegradable and biosafe nanomaterials that were demonstrated to be applicable to the delivery of nucleic acids into plant cells. This review covers studies the object of which is the plant cell and the cargo for the nanocarrier is either DNA or RNA. The following nanoplatforms that could be potentially used for nucleic acid foliar delivery via spraying are discussed: mesoporous silica nanoparticles, layered double hydroxides (nanoclay), carbon-based materials (carbon dots and single-walled nanotubes), chitosan and, finally, cell-penetrating peptides (CPPs). Hybrid nanomaterials, for example, chitosan- or CPP-functionalized carbon nanotubes, are taken into account. The selected nanocarriers are analyzed according to the following aspects: biosafety, adjustability for the particular cargo and task (e.g., organelle targeting), penetration efficiency and ability to protect nucleic acid from environmental and cellular factors (pH, UV, nucleases, etc.) and to mediate the gradual and timely release of cargo. In addition, we discuss the method of application, experimental system and approaches that are used to assess the efficiency of the tested formulation in the overviewed studies. This review presents recent progress in developing the most promising nanoparticle-based materials that are applicable to both laboratory experiments and field applications.

mRNA therapy has shown great potential in infectious disease vaccines, cancer immunotherapy, protein replacement therapy, gene editing, and other fields due to its central role in all life processes. However, mRNA is challenging to pass through the cell membrane due to its significant negative charges and degradation from RNase, so the key to mRNA therapy is efficient packaging and delivery of it with appropriate vectors. Presently researchers have developed various vectors such as viruses and liposomes, but these conventional vectors are now difficult to meet the growing requirement like safety, efficiency, and targeting, so many novel delivery vectors with unique advantages have emerged recently. This review mainly introduces two categories of novel vectors: biomacromolecules and inorganic nanoparticles, as well as two novel methods of control and administration based on these novel vectors: controlled-release administration and non-invasive administration. These novel delivery strategies have the advantages of high safety, biocompatibility, versatility, intelligence, and targeting. This paper analyzes the challenges faced by the field of mRNA delivery in depth, and discusses how to use the characteristics of novel vectors and administrations to solve these problems.

Energetics of chitosan (CS) polyplexes and conformational stability of bound DNA were studied at pH 5.0 by ITC and HS-DSC, respectively. The CS-DNA binding isotherm was well approximated by the McGhee-von Hippel model suggesting the binding mechanism to be a cooperative attachment of interacting CS ligands to the DNA matrix. Melting thermograms of polyplexes revealed the transformation of different conformational forms of bound DNA in dependence on the CS/DNA weight ratio rw. At 0 Collapse

Cancer is an intricate disease that develops as a response to a combination of hereditary and environmental risk factors, which then result in a variety of changes to the genome. The cluster of differentiation (CD44) is a type of transmembrane glycoprotein that serves as a potential biomarker for cancer stem cells (CSC) and viable targets for therapeutic intervention in the context of cancer therapy. Hyaluronic acid (HA) is a linear polysaccharide that exhibits a notable affinity for the CD44 receptor. This characteristic renders it a promising candidate for therapeutic interventions aimed at selectively targeting CD44-positive cancer cells. Treating cancer via non-viral vector-based gene delivery has changed the notion of curing illness through the incorporation of therapeutic genes into the organism. The objective of this review is to provide an overview of various hyaluronic acid-modified lipoplexes and polyplexes as potential drug delivery methods for specific forms of cancer by effectively targeting CD44.

Cancer is a major health concern worldwide and is still in a continuous surge of seeking for effective treatments. Since the discovery of RNAi and their mechanism of action, it has shown promises in targeted therapy for various diseases including cancer. The ability of RNAi to selectively silence the carcinogenic gene makes them ideal as cancer therapeutics. Oral delivery is the ideal route of administration of drug administration because of its patients' compliance and convenience. However, orally administered RNAi, for instance, siRNA, must cross various extracellular and intracellular biological barriers before it reaches the site of action. It is very challenging and important to keep the siRNA stable until they reach to the targeted site. Harsh pH, thick mucus layer, and nuclease enzyme prevent siRNA to diffuse through the intestinal wall and thereby induce a therapeutic effect. After entering the cell, siRNA is subjected to lysosomal degradation. Over the years, various approaches have been taken into consideration to overcome these challenges for oral RNAi delivery. Therefore, understanding the challenges and recent development is crucial to offer a novel and advanced approach for oral RNAi delivery. Herein, we have summarized the delivery strategies for oral delivery RNAi and recent advancement towards the preclinical stages.

Gene therapy holds great promise for treating a multitude of inherited and acquired diseases by delivering functional genes, comprising DNA or RNA, into targeted cells or tissues to elicit manipulation of gene expression. However, the clinical implementation of gene therapy remains substantially impeded by the lack of safe and efficient gene delivery vehicles. This review comprehensively outlines the novel fastest-growing and efficient non-viral gene delivery vectors, which include liposomes and lipid nanoparticles (LNPs), highly branched poly(β-amino ester) (HPAE), single-chain cyclic polymer (SCKP), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimine (PEI). Particularly, we discuss the research progress, potential development directions, and remaining challenges. Additionally, we provide a comprehensive overview of the currently approved non-viral gene therapeutics, as well as ongoing clinical trials. With advances in biomedicine, molecular biology, materials science, non-viral gene vectors play an ever-expanding and noteworthy role in clinical gene therapy.

Mobile sensing enabled by MS² technology, which integrates microfluidic and smartphone components, has seen many applications in recent years. In this direction, we developed an MS² platform (an integrated kit) for nucleic acid assay, which included a chitosan-modified minimalistic microfluidic chip (CM³-chip), a smartphone-based fluorescence detector (SF-detector), an APP for imaging and analysis, reagents, and accessories. Once the lysed sample was loaded into the CM³-chip modified by 1% concentration and 200-260 kDa molecular weight of chitosan, the following assay can be completed in approximately 1 h. The Mkit can detect 3 × 10° copies μL^-1 of plasmid DNA and its polymerase chain reaction (PCR) efficiency was 96.8%. The CM³-chip equipped for the Mkit can enrich nucleic acid from the pH = 5 of lysis buffer, instead of using conventional adsorption mediums such as the magnetic beads and silica gel membranes, which could result in unexpected impurity residuals and tedious cleaning operations. In addition, the performance of the Mkit equipped with the pristine chip was demonstrated to perform poorer than that coupled with the CM³-chip in which the enriched nucleic acid can be all used for "in-situ PCR". The universality, selectivity, and user-friendliness of the Mkit were also validated. We finally demonstrated the feasibility of the Mkit for testing artificially prepared infected samples. H5N6 and IAV-infected saliva samples provided the limits of detection of 5 × 10² copies mL^-1 and 3.24 × 10² copies mL^-1 per chamber, respectively. The streamlined assay and compact device should enable the great potential of the Mkit in research and potential diagnostic uses.

Chitosan is extensively studied as a carrier for gene delivery and is an attractive non-viral gene vector owing to its polycationic, biodegradable, and biocompatible nature. Thus, it is essential to understand the chemistry of self-assembled chitosan-DNA complexation and their structural and functional properties, enabling the formation of an effective non-viral gene delivery system. In this study, two parent chitosans (samples NAS-032 and NAS-075; Mw range ~118-164 kDa) and their depolymerised derivatives (deploy nas-032 and deploy nas-075; Mw range 6-14 kDa) with degrees of acetylation 43.4 and 4.7%, respectively, were used to form polyelectrolyte complexes (PECs) with DNA at varying [-NH3+]/[-PO4-] (N/P) molar charge ratios. We investigated the formation of the PECs using ζ-potential, asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS), refractive index (RI), ultraviolet (UV) and dynamic light scattering (DLS) detectors, and TEM imaging. PEC formation was confirmed by ζ-potential measurements that shifted from negative to positive values at N/P ratio ~2. The radius of gyration (Rg) was determined for the eluting fractions by AF4-MALS-RI-UV, while the corresponding hydrodynamic radius (Rh), by the DLS data. We studied the influence of different cross-flow rates on AF4 elution patterns for PECs obtained at N/P ratios 5, 10, and 20. The determined rho shape factor (ρ = Rg/Rh) values for the various PECs corresponded with a sphere morphology (ρ ~0.77-0.85), which was consistent with TEM images. The results of this study represent a further step towards the characterisation of chitosan-DNA PECs by the use of multi-detection AF4 as an important tool to fractionate and infer aspects of their morphology.

Zoledronic acid (ZA) is known as a potent bisphosphonate in osteogenic differentiation, but at high doses, it possesses toxic effects and causes decreased proliferation and differentiation of osteoblasts. Therefore, encapsulation of ZA into nanoparticles and control of its release is expected to promote differentiation of stem cells into osteoblasts. The present work aimed to develop a simple method for synthesis of monodisperse ZA-loaded chitosan (CS) nanoparticles. In this regard, we proposed a microfluidic synthesis of nanoparticles through the ionic cross-linking of CS in the presence of ZA without a crosslinker. The main advantages of these microfluidic generated nanoparticles were narrow size distribution and fine spherical shape. Conversely, the nanoparticles that were synthesized using a bulk mixing method had an irregular shape with a broad size distribution. Real-time PCR assay as well as alizarin red staining were used to evaluate the in-vitro osteogenic potential of the nanoparticles. The results indicated that the controlled release of ZA from the microfluidic system generated uniform nanoparticles, improving the osteogenic differentiation of mesenchymal stem cells. Additionally, this microfluidic device provided the well-controlled synthesis of novel nanoparticles with a modified CS macromolecular polymer for targeted drug delivery systems.

The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.

Biological characteristics of natural polymers make microbial polysaccharides an excellent choice for biopharmaceuticals. Due to its easy purifying procedure and high production efficiency, it is capable of resolving the existing application issues associated with some plant and animal polysaccharides. Furthermore, microbial polysaccharides are recognized as prospective substitutes for these polysaccharides based on the search for eco-friendly chemicals. In this review, the microstructure and properties of microbial polysaccharides are utilized to highlight their characteristics and potential medical applications. From the standpoint of pathogenic processes, in-depth explanations are provided on the effects of microbial polysaccharides as active ingredients in the treatment of human diseases, anti-aging, and drug delivery. In addition, the scholarly developments and commercial applications of microbial polysaccharides as medical raw materials are also discussed. The conclusion is that understanding the use of microbial polysaccharides in biopharmaceuticals is essential for the future development of pharmacology and therapeutic medicine.

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL^-1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.

Chitosan derived from chitin gas gathered much interest as a biopolymer due to its known and possible broad applications. Chitin is a nitrogen-enriched polymer abundantly present in the exoskeletons of arthropods, cell walls of fungi, green algae, and microorganisms, radulae and beaks of molluscs and cephalopods, etc. Chitosan is a promising candidate for a wide variety of applications due to its macromolecular structure and its unique biological and physiological properties, including solubility, biocompatibility, biodegradability, and reactivity. Chitosan and its derivatives have been known to be applicable in medicine, pharmaceuticals, food, cosmetics, agriculture, the textile and paper industries, the energy industry, and industrial sustainability. More specifically, their use in drug delivery, dentistry, ophthalmology, wound dressing, cell encapsulation, bioimaging, tissue engineering, food packaging, gelling and coating, food additives and preservatives, active biopolymeric nanofilms, nutraceuticals, skin and hair care, preventing abiotic stress in flora, increasing water availability in plants, controlled release fertilizers, dye-sensitised solar cells, wastewater and sludge treatment, and metal extraction. The merits and demerits associated with the use of chitosan derivatives in the above applications are elucidated, and finally, the key challenges and future perspectives are discussed in detail.

The use of RNA interference mechanism and small interfering RNA (siRNA) in cancer gene therapy is a very promising approach. However, the success of gene silencing is underpinned by the efficient delivery of intact siRNA into the targeted cell. Nowadays, chitosan is one of the most widely studied non-viral vectors for siRNA delivery, since it is a biodegradable, biocompatible and positively charged polymer able to bind to the negatively charged siRNA forming nanoparticles (NPs) that will act as siRNA delivery system. However, chitosan shows several limitations such as low transfection efficiency and low solubility at physiological pH. Therefore, a variety of chemical and non-chemical structural modifications of chitosan were investigated in the attempt to develop a chitosan derivative showing the features of an ideal siRNA carrier. In this review, the most recently proposed chemical modifications of chitosan are outlined. The type of modification, chemical structure, physicochemical properties, siRNA binding affinity and complexation efficiency of the modified chitosan are discussed. Moreover, the resulting NPs characteristics, cellular uptake, serum stability, cytotoxicity and gene transfection efficiency in vitro and/or in vivo are described and compared to the unmodified chitosan. Finally, a critical analysis of a selection of modifications is included, highlighting the most promising ones for this purpose in the future.

Due to recalcitrant microangiopathy and chronic infection, traditional treatments do not easily produce satisfactory results for chronic diabetic ulcers. In recent years, due to the advantages of high biocompatibility and modifiability, an increasing number of hydrogel materials have been applied to the treatment of chronic wounds in diabetic patients. Research on composite hydrogels has received increasing attention since loading different components can greatly increase the ability of composite hydrogels to treat chronic diabetic wounds. This review summarizes and details a variety of newly loaded components currently used in hydrogel composites for the treatment of chronic diabetic ulcers, such as polymer/polysaccharides/organic chemicals, stem cells/exosomes/progenitor cells, chelating agents/metal ions, plant extracts, proteins (cytokines/peptides/enzymes) and nucleoside products, and medicines/drugs, to help researchers understand the characteristics of these components in the treatment of diabetic chronic wounds. This review also discusses a number of components that have not yet been applied but have the potential to be loaded into hydrogels, all of which play roles in the biomedical field and may become important loading components in the future. This review provides a "loading component shelf" for researchers of composite hydrogels and a theoretical basis for the future construction of "all-in-one" hydrogels.

The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency. In this work, the pH-responsiveness of the poly(2-(diisopropylamino)ethyl methacrylate)) (PDPA) block was investigated for the encapsulation and delivery of small RNAs (sRNA) to cancer cells. The pH responsiveness was dependent on the protonation profile of the tertiary amines of PDPA, which directly affected the electrostatic interactions established with RNA. Thus, block copolymers based on poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA) and PDPA, POEOMA-b-PDPA, were synthesized by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP). The structure of the block copolymers was characterized by size exclusion chromatography and ¹H NMR spectroscopy. The copolymers allowed effective complexation of model sRNAs and a pre-miRNA with efficiencies of about 89 % and 91 %, respectively. The characterization by dynamic light scattering revealed that these systems had sizes between 76 and 1375 nm. It was also found that the morphology of the polyplexes depended on the pH, since the preparation at a pH lower than the pKa of the copolymers resulted in spherical but polydisperse particles, while higher pH values resulted in nanoparticles with more homogeneous size, but altered morphology. Moreover, due to pH-responsiveness, it was achieved the release of RNA at pH higher than the pKa of the copolymers, while maintaining its integrity. The polyplexes also showed a high potential to protect RNA from RNases. The transfection of a lung cancer model (A549) and fibroblast cell lines showed that these polyplexes did not cause cell toxicity. In addition, the polyplexes enabled the effective transfection of the A549 cell line with pre-miRNA-29b and miRNA-29b, resulting in a decrease of expression levels of the target DNMT3B gene by approximately 51 % and 47 %, respectively. Overall, the POEOMA-b-PDPA copolymers proved to be a promising strategy for developing responsive delivery systems, that can play a critical role in some diseases, such as cancer, where pH varies between the intra and extracellular environments.

Epithelial-mesenchymal transition (EMT) is the culprit of tumor invasion and metastasis. As a critical transcription factor that induces EMT, snail is of great importance in tumor progression, and knocking down its expression by small interfering RNA (siRNA) may inhibit tumor metastasis. Herein, we developed a core-shelled bioinspired low-density lipoprotein (bio-LDL) in which snail siRNA-loaded calcium phosphate nanoparticles were wrapped as the core and doxorubicin was embedded in the outer phospholipids modified with a synthetic peptide of apoB100 targeting LDL receptor-abundant tumor cells. Bio-LDL exhibited pH-responsive release, lysosomal escape ability, enhanced cytotoxicity and apoptotic induction. Bio-LDL could significantly inhibit the expression of snail and regulate EMT-related proteins to reduce tumor migration and invasion in vitro. Bio-LDL also displayed favorable tumor targeting and synergistic inhibition of tumor growth and metastasis in vivo. Therefore, the multifunctional bio-LDL will be a promising co-delivery vector and holds potential value for clinical translation.