Focused ultrasound on the substantia nigra enables safe neurotensin-polyplex nanoparticle-mediated gene delivery to dopaminergic neurons intranasally and by blood circulation.

Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson's disease because they do not cross the blood-brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 µL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson's disease.

Superior COL7A1 and TGM1 gene expression in difficult-to-transfect skin cell mediated by highly branched poly(ß-amino esters) through stepwise fractionation.

Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with Mw ranging from 7.6 to 131.8 kg/mol and D < 2.0 from the one crude HPAE23.7k, and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE20.6k, exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100%), whereas polymer E100 demonstrated dose-dependent behaviour (20-90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42% at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Impact of Linker Groups on Self-Assembly, Gene Transfection, Antibacterial Activity, and In Vitro Cytotoxicity of Cationic Bolaamphiphiles.

Cationic bolaamphiphiles have gained significant attention in various research fields, including materials science, drug delivery, and gene therapy, due to their unique properties and potential applications. The objective of the current research is to develop more effective cationic bolaamphiphiles. Thus, we have designed and synthesized two cationic bolaamphiphiles (-(CH2)12(2,3-dihydroxy-N,N-dimethyl-N-(3-ureidopropyl)propan-1-aminium chloride))2 (C12(DDUPPAC)2)) and (-(CH2)12(N-(3-(carbamoyloxy)propyl)-2,3-dihydroxy-N,N-dimethylpropan-1-aminium chloride)2 (C12(CPDDPAC)2) containing urea and urethane linkages, respectively. We have investigated their self-assembly properties in water using several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. Their biological applications, e.g., in vitro gene transfection, antibacterial activity, and cytotoxicity, were studied. Both bolaamphiphiles were observed to produce aggregates larger than spherical micelles above a relatively low critical aggregation concentration (cac). The calorimetric experiments suggested the thermodynamically favorable spontaneous aggregation of both bolaforms in water. The results of interaction studies led to the conclusion that C12(CPDDPAC)2 binds DNA with a greater affinity than C12(DDUPPAC)2. Also, C12(CPDDPAC)2 is found to act as a more efficient gene transfection vector than C12(DDUPPAC)2 in 264.7 cell lines. The in vitro cytotoxicity assay using MTT, however, revealed that neither of the bolaamphiphiles was toxic, even at higher quantities. Additionally, both bolaforms show beneficial antibacterial activity.

TPGS-b-PBAE Copolymer-Based Polyplex Nanoparticles for Gene Delivery and Transfection In Vivo and In Vitro.

Poly (ß-amino ester) (PBAE) is an exceptional non-viral vector that is widely used in gene delivery, owing to its exceptional biocompatibility, easy synthesis, and cost-effectiveness. However, it carries a high surface positive charge that may cause cytotoxicity. Therefore, hydrophilic d-α-tocopherol polyethylene glycol succinate (TPGS) was copolymerised with PBAE to increase the biocompatibility and to decrease the potential cytotoxicity of the cationic polymer-DNA plasmid polyplex nanoparticles (NPs) formed through electrostatic forces between the polymer and DNA. TPGS-b-PBAE (TBP) copolymers with varying feeding molar ratios were synthesised to obtain products of different molecular weights. Their gene transfection efficiency was subsequently evaluated in HEK 293T cells using green fluorescent protein plasmid (GFP) as the model because free GFP is unable to easily pass through the cell membrane and then express as a protein. The particle size, ζ-potential, and morphology of the TBP2-GFP polyplex NPs were characterised, and plasmid incorporation was confirmed through gel retardation assays. The TBP2-GFP polyplex NPs effectively transfected multiple cells with low cytotoxicity, including HEK 293T, HeLa, Me180, SiHa, SCC-7 and C666-1 cells. We constructed a MUC2 (Mucin2)-targeting CRISPR/cas9 gene editing system in HEK 293T cells, with gene disruption supported by oligodeoxynucleotide (ODN) insertion in vitro. Additionally, we developed an LMP1 (latent membrane protein 1)-targeting CRISPR/cas9 gene editing system in LMP1-overexpressing SCC7 cells, which was designed to cleave fragments expressing the LMP1 protein (related to Epstein-Barr virus infection) and thus to inhibit the growth of the cells in vivo. As evidenced by in vitro and in vivo experiments, this system has great potential for gene therapy applications.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

Development of triple-functionalized calcium phosphate nanoparticles as an advanced drug delivery system for bone tissue repair.

Introduction: During tissue repair or regeneration, several bioactive molecules are released and interact with each other and act as complex additives or inhibitors for tissue reconstruction. In this study, the bone-healing effects of the combination treatment with tumor necrosis factor-α (TNF-α) inhibition, vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein-7 (BMP-7) release by gene silencing, and gene transfection with calcium phosphate nanoparticles (CaP) in the rat femoral head was histologically, morphologically, and biochemically evaluated. Methods: A triple-functionalized paste of CaP carrying plasmid DNA encoding for BMP-7 and for VEGF), and siRNA against TNF-α was developed and denoted as CaP3mix. To compare the effects of 3mixCaP, CaP with plasmid DNA encoding BMP-7, VEGF, or siRNA encoding TNF-α was prepared and denoted as CaP/PEI/pBMP-7/SiO2, CaP/PEI/pVEGF/SiO2, or CaP/PEI/siRNA-TNF-α/SiO2, respectively. The bone healing in bone defects in the rat femoral head was investigated after 10 and 21 days of implantation. Results: The levels of bone formation-related markers OCN, Runx2, and SP7 increased at the protein and gene levels in 3mixCaP after 10 days, and 3mixCaP significantly accelerated bone healing compared with the other treatments after 21 days of implantation. Conclusion: The triple-functionalized CaP paste loading plasmid DNA encoding BMP-7 and VEGF and siRNA encoding TNF-α is a promising bioactive material for bone tissue repair.

Nano-Plumber Reshapes Glymphatic-Lymphatic System to Sustain Microenvironment Homeostasis and Improve Long-Term Prognosis after Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Long-term changes in the microenvironment of the brain contribute to the degeneration of neurological function following TBI. However, current research focuses primarily on short-term modulation during the early phases of TBI, not on the critical significance of long-term homeostasis in the brain microenvironment. Notably, dysfunction of the glymphatic-lymphatic system results in the accumulation of danger/damage-associated molecular patterns (DAMPs) in the brain, which is regarded as the leading cause of long-term microenvironmental disturbances following TBI. Here, a nanostructure, Nano-plumber, that co-encapsulates the microenvironment regulator pro-DHA and the lymphatic-specific growth factor VEGF-C is developed, allowing for a sustainable and orderly regulation of the microenvironment to promote long-term neurological recovery. Nano-plumber reverses the injury microenvironment by suppressing microglia and astrocytes activation and maintaining reduced activation via enhanced glymphatic-lymphatic drainage, and significantly improves the neurological function of rodents with TBI. This study demonstrates that glymphatic-lymphatic system reconstruction is essential for enhancing long-term prognosis following TBI, and that the Nano-plumber developed here may serve as a clinically translatable treatment option for TBI.

Enhancing the Gene Transfection of Poly(ß-amino ester)/DNA Polyplexes by Modular Manipulation of Amphiphilicity.

Poly(ß-amino ester)s (PAEs) have been widely developed for gene delivery, and hydrophobic modification can further enhance their gene transfection efficiency. However, systematic manipulation of amphiphilicity of PAEs through copolymerization with hydrophobic monomers is time-consuming and, to some extent, uncontrollable. Here, a modular strategy is developed to manipulate the amphiphilicity of the PAE/DNA polyplexes. A hydrophobic polymer (DD-C12-122) and a hydrophilic polymer (DD-90-122) are synthesized separately and used as a hydrophobic module and a hydrophilic module, respectively. The amphiphilicity of polyplexes could be manipulated by changing the ratio of the hydrophobic module and hydrophilic module. Using the modular strategy, the PAE/DNA polyplexes with the highest gene transfection efficiency and safety profile as well as possible mechanisms are identified. The modular strategy provides a novel way to engineer the hydrophobicity of PAEs to improve their gene transfection and can be easily generalized and potentially extended to other polymeric gene delivery systems.

Therapeutic Effects and Underlying Mechanism of SOCS-com Gene-Transfected ADMSCs in Pressure Ulcer Mouse Models.

Although the proportion of ulcer patients with medical problems among the elderly has increased with the extension of human life expectancy, treatment efficiency is drastically low, incurring substantial social costs. MSCs have independent regeneration potential, making them useful in clinical trials of difficult-to-treat diseases. In particular, ADMSCs are promising in the stem cell therapy industry as they can be obtained in vast amounts using non-invasive methods. Furthermore, studies are underway to enhance the regeneration potential of ADMSCs using cytokines, growth factors, and gene delivery to generate highly functional ADMSCs. In this study, key regulators of wound healing, SOCS-1, -3, and -5, were combined to maximize the regenerative potential of ADMSCs in pressure ulcer treatments. After transfecting SOCS-1, -3, -5, and SOCS-com into ADMSCs using a non-viral method, the expression of the inflammatory factors TNF-alpha, INF-gamma, and IL-10 was confirmed. ADMSCs transfected with SOCS-com showed decreased overall expression of inflammatory factors and increased expression of anti-inflammatory factors. Based on these results, we implanted ADMSCs transfected with SOCS-com into a pressure ulcer mouse model to observe their subsequent wound-healing effects. Notably, SOCS-com improved wound closure in ulcers, and reconstruction of the epidermis and dermis was observed. The healing mechanism of ADMSCs transfected with SOCS-com was examined by RNA sequencing. Gene analysis results confirmed that expression changes occurred in genes of key regulators of wound healing, such as chemokines, MMP-1, 9, CSF-2, and IL-33, and that such genetic changes enhanced wound healing in ulcers. Based on these results, we demonstrate the potential of ADMSCs transfected with SOCS-com as an ulcer treatment tool.

Peritoneal Gene Transfection of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand for Tumor Surveillance and Prophylaxis.

Peritoneal metastasis is very common in gastrointestinal, reproductive, and genitourinary tract cancers in late stages or postsurgery, causing poor prognosis, so effective and nontoxic prophylactic strategies against peritoneal metastasis are highly imperative. Herein, we demonstrate the first gene transfection as a nontoxic prophylaxis preventing peritoneal metastasis or operative metastatic dissemination. Lipopolyplexes of TNF-related-apoptosis-inducing-ligand (TRAIL) transfected peritonea and macrophages to express TRAIL for over 15 days. The expressed TRAIL selectively induced tumor cell apoptosis while exempting normal tissue, providing long-term tumor surveillance. Therefore, tumor cells inoculated in the pretransfected peritoneal cavity quickly underwent apoptosis and, thus, barely formed tumor nodules, significantly prolonging the mouse survival time compared with chemotherapy prophylaxis. Furthermore, lipopolyplex transfection showed no sign of toxicity. Therefore, this peritoneal TRAIL-transfection is an effective and safe prophylaxis, preventing peritoneal metastasis.

Lipoic Acid-Based Poly(disulfide)s as Versatile Biomolecule Delivery Vectors and the Application in Tumor Immunotherapy.

Intracellular delivery of therapeutic biomacromolecules, including nucleic acids and proteins, attracts extensive attention in biotherapeutics for various diseases. Herein, a strategy is proposed for the construction of poly(disulfide)s for the efficient delivery of both nucleic acids and proteins into cells. A convenient photo-cross-linking polymerization was adopted between disulfide bonds in two modified lipoic acid monomers (Zn coordinated with dipicolylamine analogue (ZnDPA) and guanidine (GUA)). The disulfide-containing main chain of the resulting poly(disulfide)s was responsive to reducing circumstance, facilitating the release of cargos. By screening the feeding ratio of ZnDPA and GUA, the resulting poly(disulfide)s exhibited better performance in the delivery of nucleic acids including plasmid DNA and siRNA than commercially available transfection reagents. Cellular uptake results revealed that the polymer/cargo complexes entered the cells mainly following a thiol-mediated uptake pathway. Meanwhile, the polymer could also efficiently deliver proteins into cells without an obvious loss of protein activity, showing the versatility of the poly(disulfide)s for the delivery of various biomacromolecules. Moreover, the in vivo therapeutic effect of the materials was verified in the E.G7-OVA tumor-bearing mice. Ovalbumin-based nanovaccine induced a strong cellular immune response, especially cytotoxic T lymphocyte cellular immune response, and inhibited tumor growth. These results revealed the promise of the poly(disulfide)s in the application of both gene therapy and immunotherapy.

Dendrimers: Advancements and Potential Applications in Cancer Diagnosis and Treatment-An Overview.

Cancer is a leading cause of death worldwide, and the main treatment methods for this condition are surgery, chemotherapy, and radiotherapy. These treatment methods are invasive and can cause severe adverse reactions among organisms, so nanomaterials are increasingly used as structures for anticancer therapies. Dendrimers are a type of nanomaterial with unique properties, and their production can be controlled to obtain compounds with the desired characteristics. These polymeric molecules are used in cancer diagnosis and treatment through the targeted distribution of some pharmacological substances. Dendrimers have the ability to fulfill several objectives in anticancer therapy simultaneously, such as targeting tumor cells so that healthy tissue is not affected, controlling the release of anticancer agents in the tumor microenvironment, and combining anticancer strategies based on the administration of anticancer molecules to potentiate their effect through photothermal therapy or photodynamic therapy. The purpose of this review is to summarize and highlight the possible uses of dendrimers regarding the diagnosis and treatment of oncological conditions.

Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

A transfection vector based on a peptide dendrimer (1) has been developed and its abilities for DNA binding and transport have been investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector 1* condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken up by the endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.

A New Optimization Strategy of Highly Branched Poly(ß-Amino Ester) for Enhanced Gene Delivery: Removal of Small Molecular Weight Components.

Highly branched poly(ß-amino ester) (HPAE) has become one of the most promising non-viral gene delivery vector candidates. When compared to other gene delivery vectors, HPAE has a broad molecular weight distribution (MWD). Despite significant efforts to optimize HPAE targeting enhanced gene delivery, the effect of different molecular weight (MW) components on transfection has rarely been studied. In this work, a new structural optimization strategy was proposed targeting enhanced HPAE gene transfection. A series of HPAE with different MW components was obtained through a stepwise precipitation approach and applied to plasmid DNA delivery. It was demonstrated that the removal of small MW components from the original HPAE structure could significantly enhance its transfection performance (e.g., GFP expression increased 7 folds at w/w of 10/1). The universality of this strategy was proven by extending it to varying HPAE systems with different MWs and different branching degrees, where the transfection performance exhibited an even magnitude enhancement after removing small MW portions. This work opened a new avenue for developing high-efficiency HPAE gene delivery vectors and provided new insights into the understanding of the HPAE structure-property relationship, which would facilitate the translation of HPAEs in gene therapy clinical applications.

Toward Pathogenic Biofilm Suppressors: Synthesis of Amino Derivatives of Pillar[5]arene and Supramolecular Assembly with DNA.

New amino derivatives of pillar[5]arene were obtained in three stages with good yields. It was shown that pillar[5]arene containing thiaether and tertiary amino groups formed supramolecular complexes with low molecular weight model DNA. Pillar[5]arene formed complexes with a DNA nucleotide pair at a ratio of 1:2 (macrocycle/DNA base pairs), as demonstrated by UV-visible and fluorescence spectroscopy. The association constants of pillar[5]arene with DNA were lgKass1:1 = 2.38 and lgKass1:2 = 5.07, accordingly. By using dynamic light scattering and transmission electron microscopy, it was established that the interaction of pillar[5]arene containing thiaether and tertiary amino groups (concentration of 10-5 M) with a model nucleic acid led to the formation of stable nanosized macrocycle/DNA associates with an average particle size of 220 nm. It was shown that the obtained compounds did not exhibit a pronounced toxicity toward human adenocarcinoma cells (A549) and bovine lung epithelial cells (LECs). The hypothesis about a possible usage of the synthesized macrocycle for the aggregation of extracellular bacterial DNA in a biofilm matrix was confirmed by the example of St. Aureus. It was found that pillar[5]arene at a concentration of 10-5 M was able to reduce the thickness of the St. Aureus biofilm by 15%.

Drug resistance of hepatoma cells induced by ATP­binding cassette transporter G2 by reducing intracellular drug concentration.

The side effects and drug resistance during chemotherapy seriously affect the outcome of and may lead to the failure of chemotherapy for patients with hepatoma. The aim of the present study was to investigate the association between the expression of ATP-binding cassette transporter G2 (ABCG2) in hepatoma cells and the drug resistance of hepatoma. An MTT assay was used to determine the half-maximal inhibitory concentration (IC50) of Adriamycin (ADM) in hepatoma HepG2 cells after treatment with ADM for 24 h. An ADM-resistant hepatoma cell subline, HepG2/ADM, was generated from the HepG2 hepatoma cell line through a stepwise selection with ADM doses from 0.01 to 0.1 µg/ml. The HepG2/ABCG2 cell line, an ABCG2-overexpressing hepatoma cell line, was established by transfecting the ABCG2 gene into HepG2 cells. The MTT assay was then used to detect the IC50 of ADM in HepG2/ADM and HepG2/ABCG2 cells after treatment with ADM for 24 h and the resistance index was calculated. The apoptosis, cell cycle and ABCG2 protein expression levels in HepG2/ADM, HepG2/ABCG2 cells, HepG2/PCDNA3.1 and their parental HepG2 cells were detected by flow cytometry. In addition, flow cytometry was used to detect the efflux effect of HepG2/ADM and HepG2/ABCG2 cells after ADM treatment. ABCG2 mRNA expression in cells was detected by reverse transcription-quantitative PCR. After 3 months of ADM treatment, HepG2/ADM cells grew stably in the cell culture medium containing 0.1 µg/ml ADM and the cells were named HepG2/ADM cells. ABCG2 was overexpressed in HepG2/ABCG2 cells. The IC50 of ADM in HepG2, HepG2/PCDNA3.1, HepG2/ADM and HepG2/ABCG2 cells was 0.72±0.03, 0.74±0.01, 11.17±0.59 and 12.75±0.47 µg/ml, respectively. The cell apoptotic rate of HepG2/ADM and HepG2/ABCG2 cells was not significantly different compared with that of HepG2 and HepG2/PCDNA3.1 cells (P>0.05), but the G0/G1 phase population of the cell cycle decreased and the proliferation index increased significantly (P<0.05). The expression levels of ABCG2 gene and protein in HepG2/ADM and HepG2/ABCG2 cells were significantly higher than those in HepG2 and HepG2/PCDNA3.1 cells (P<0.01), but there was no significant difference between HepG2 and HepG2/PCDNA3.1 cells (P>0.05). The ADM efflux effect of HepG2/ADM and HepG2/ABCG2 cells was significantly higher than that of parental HepG2 and HepG2/PCDNA3.1 cells (P<0.05). Therefore, the present study demonstrated that ABCG2 expression is highly increased in drug-resistant hepatoma cells and that high expression of ABCG2 is involved in the drug resistance of hepatoma by reducing the intracellular drug concentration.

Introduction of tenomodulin by gene transfection vectors for rat bone tissue regeneration.

Introduction: Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique in vitro and in vivo was investigated in this study. Methods: Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Results: Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9 ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Conclusions: Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.