Assessment of current practices and perceived effectiveness of injectable polynucleotide for enlarged facial pores among cosmetic physicians: A survey-based evaluation.

BACKGROUND: Polynucleotides stimulate collagen formation and are used clinically to enhance elasticity. In this study, we investigated current practices and perceived effectiveness of polynucleotide injection treatment for enlarged facial pores among cosmetic physicians. MATERIALS AND METHODS: A survey was developed to investigate clinicians' use and effectiveness of polynucleotides in the treatment of enlarged facial pores. This survey was distributed to clinicians at the Korean Aesthetic Surgery & Laser Society Autumn Symposium. RESULTS: A total of 407 physicians who used polynucleotides for enlarged facial pores were enrolled in the survey. Polynucleotides were used by 75.7%, 87.7%, and 72.2% of physicians for enlarged facial pores caused by excessive sebum production, reduced elasticity, and acne, respectively. Among those users, 81.4%, 83.8%, and 76.8% in those same categories, respectively, responded that polynucleotides were "very effective" or "effective." Furthermore, most clinicians combined polynucleotides with microneedle radiofrequency as energy-based devices and with botulinum toxin as injection therapy. CONCLUSION: This study highlights the widespread use and perceived efficacy of polynucleotide injection among cosmetic physicians in the Republic of Korea for enlarged facial pores due to excessive sebum production, reduced elasticity, and acne. Positive feedback from practitioners supports the benefits of using polynucleotides in enlarged facial pore treatment.

Inflammation enhances the vaccination potential of CD40-activated B cells in mice.

Vaccination with antigen-pulsed CD40-activated B (CD40-B) cells can efficiently lead to the in vivo differentiation of naive CD8+ T cells into fully functional effectors. In contrast to bone marrow-derived dendritic cell (BMDC) vaccination, CD40-B cell priming does not allow for memory CD8+ T-cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40-B cells induce lower expression of several genes regulated by T-cell receptor signaling, costimulation, and inflammation (signals 1-3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40-B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8+ T cells as compared to BMDCs. Furthermore, CD40-B cells produce less inflammatory mediators, such as IL-12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic-polyribocytidylic acid with CD40-B-cell immunization allowed for the generation of long-lived and functional CD8+ memory T cells. In conclusion, it is possible to manipulate CD40-B-cell vaccination to promote the formation of long-lived functional CD8+ memory T cells, a key step before translating the use of CD40-B cells for therapeutic vaccination.

Long-chain polynucleotide filler for skin rejuvenation: efficacy and complications in five patients.

Aging well has become the new target of preventative medicine, and aesthetic dermatology can contribute to this request. The polynucleotide (PN) containing products not only fill the space, but improve tissue regeneration, resulting in more natural tissue regeneration. Five Korean women received four times injections of long-chain PN filler in two-week intervals for skin rejuvenation. About 0.05 mL of material was injected in 40 points of one-side cheek. The pore and skin thickness were markedly improved in the patients in their 30s, whereas skin tone, melanin, wrinkles, and sagging were noticeably improved for patients in their 40s. There are no serious side effects. In conclusion, intradermal long-chain PN filler injection seems to be an effective and safe treatment for skin rejuvenation.

Trophic effects of polynucleotides and hyaluronic acid in the healing of venous ulcers of the lower limbs: a clinical study.

The aim of this study was to evaluate the results of treatment of venous lower limbs ulcers through the topical application of polynucleotides and hyaluronic acid gel (PNHA): Nucliaskin S™ (Mastelli srl, San Remo, Italy). This study was carried out in 39 consecutive patients who were randomly allocated to two groups: group I (20 patients) received treatment with PNHA (topical gel application two times a week, for a total of 6 weeks); group II (19 patients) received only hyaluronic acid (HA) topical application. All patients received a surgical debridement of the ulcerative lesions before topical treatment with PNHA or HA. Pre-treatment data indicated the area of ulceration. The number of healed ulcers and the variation in area of ulceration were considered as endpoints. The endpoints were observed after 45 days from the beginning of treatment. Complete wound healing occurred in 60% of limbs of group I and in 22% of those of group II patients. The average area reduction was 67% versus 34% in patients of group I and II, respectively. No side effects were recorded in both groups. Our experience shows that PNHA has an elevated trophic effect and speeds the healing rate of venous lower limb ulcers. This treatment may be a valid option in clinical practice.

Intrarticular treatment of osteoartropaty knee with polynucleotides: a pilot study with medium-term follow-up.

Knee osteoarthritis is a major cause of disability in the elderly. Many therapies are nowadays available, ranging from non-pharmacologic to pharmacological approaches like visco-supplementation, oral supplements or topical treatments, but a flawless treatment is still to be found. Visco-supplementation represents a valid treatment option for reducing pain associated with knee osteoarthritis and improving function in the affected joint. Many literature data report on the efficacy and safety profiles of hyaluronic acid in knee osteoarthritis, however the efficacy of intra-articular hyaluronic acid remains controversial, in fact while several clinical trials claimed a disease-modifying effect for hyaluronic acid, subsequent meta-analyses have cast doubts on this fact. The ideal intra-articular treatment for osteoarthritis should not only provide a mechanical protection of the cartilage surface, but also restore condrocytes’ homeostasis by restoring the physiological articular micro-environment and supplying nutrients. In this perspective an innovative medical product made up of polynucleotides (Condrotide) has been developed. The aim of this study is to test the 2-months efficacy in pain relief and improving function of intra-articular injections of Condrotide in patients with knee osteoarthritis or with grade III or IV chondropathy. Ninety-five subjects (33 men, 62 women), aged between 53 and 80, were included between May 2011 to July 2012. All subjects received intra-articular injections of Condrotide and were evaluated with the Knee injury and Osteoarthritis Outcome Score (KOOS), the NRS scale for pain assessment, the measurement of the range of motion (R.O.M.). In all subjects a significant improvement was found in KOOS score after 60 days. The mean global NRS pain decreased in both groups and there was also a R.O.M. improvement. These results show that the intra-articular administration of nucleotides in subjects with both severe knee arthritis and chondropathy can be recommended since is able to reverse in the short and medium term symptoms and function with a significant improvement in quality of life.

Identification and molecular cloning of Atlantic cod (Gadus morhua) activating transcription factor 3 (ATF3) transcript and its induction in spleen following intraperitoneal polyriboinosinic polyribocytidylic acid injection.

Activating transcription factor 3 (ATF3) participates in cellular processes to adapt to various extra- and intra-cellular changes including the modulation of immunity to prevent uncontrolled immune responses to pathogens. In teleost fishes, the involvement of ATF3 in immune response has not been documented. In this study, the putative Atlantic cod (Gadus morhua) ATF3 transcript was identified by performing rapid amplification of cDNA ends (RACE) based on unknown expressed sequence tags (ESTs) that are potentially inducible by polyriboinosinic polyribocytidylic acid (pIC, a synthetic double-stranded RNA viral mimic) in Atlantic cod. ATF3-like ESTs were the most abundant unknown transcript (i.e. lacking significant BLAST hits) generated from a previously constructed cDNA library enriched for pIC inducible transcripts in Atlantic cod spleen. The full-length cDNA of cod ATF3 consists of 2329 nucleotides with an open reading frame (ORF) of 735 bp encoding 244 amino acids. The deduced amino acid sequence of Atlantic cod ATF3 shares over 45% identity with its putative orthologs from other vertebrates. In addition, the presence of a conserved basic region leucine zipper (bZIP) domain in the deduced Atlantic cod ATF3-like protein further supports its identity as an ATF3 homolog. In the spleen of Atlantic cod challenged with intraperitoneal (IP) injections of pIC, the time-course transcript expression of ATF3 was studied using quantitative reverse transcription-polymerase chain reaction (QPCR). At 6 h following the pIC injection, the relative expression level of ATF3 mRNA was significantly up-regulated in comparison to a pre-injected control (61.9-fold) and its time-matched saline-injected control (97.3-fold). At 24 h following the pIC injection, the mRNA expression level of cod ATF3 had subsided and was no longer significantly different from its pre-injected control, but significantly higher (1.88-fold) than its time-matched saline-injected control. Collectively, these results suggest that ATF3 may be involved in the modulation of innate anti-viral response in Atlantic cod.

Regulation of the immune system by synthetic polynucleotides. V. Effect on cell-associated immunoglobulin receptors and immunological memory.

Addition of polyadenylic-polyuridylic acid in complex form (poly A:U) without antigen to a suspension of spleen cells obtained from BALB/Aj mice primed 6 wk previously with human gamma-globulin (HGG) resulted in an immediate fourfold increase over background number of anti-HGG rosette-forming cells (RFC). Culture of similar cells in the presence of puromycin for 1-6 hr before poly A:U did not significantly reduce the response. Continued culture of primed spleen cells in the presence of poly A:U, resulted in a decrease of RFC to background levels within an hour followed by an increase again 6 hr later. This later increase in RFC was inhibited by addition of puromycin to the culture medium. The nonspecific stimulation by poly A:U of antibody production by primed spleen cells also was induced in vivo. Increases in splenic RFC were detectable 6 hr after intravenous injection of poly A:U alone, without antigen, into primed mice. The response peaked at 18 hr and had dissipated completely within 3 days. A second injection of poly A:U 24 hr or later after the first injection resulted in a second response, similar to the first with respect to kinetics and intensity. Rosette formation by poly A:U-stimulated cells could not be inhibited by mitotic poisons, but was inhibited by treatment of the cells with goat anti-mouse gamma-globulin serum, suggesting that the antibody involved was a 7S gamma-globulin. The decrease in RFC induced by culture of primed cells for 1 hr in poly A:U paralleled a decrease in secondary responsiveness of the cells to antigen. This poly A:U-induced inhibition of secondary responsiveness could be reversed by suspending the treated cells in supernatant fluids derived from poly A:U-stimulated cultures. The reversal was specific in that supernatant fluids removed from bovine serum albumin (BSA)-primed cells by poly A:U did not stimulate the response of HGG-primed cells to HGG. However supernatant fluids from BSA-primed cells caused the production of anti-HGG RFC if BSA rather than HGG was used as triggering antigen. The active factor in the supernatant fluids appeared to be a 7S gamma-globulin since activity was lost after 45 min incubation of the supernatant fluids in the presence of goat anti-mouse 7S gamma-globulin serum.

Efficacy of intra-articular polynucleotides in the treatment of knee osteoarthritis: a randomized, double-blind clinical trial.

This randomized, double-blind clinical trial was conducted over 16 weeks to assess the efficacy and safety profile of intra-articular polynucleotides gel injections in the treatment of knee osteoarthritis associated with persistent knee pain. 60 patients were enrolled and randomized to receive intra-articular polynucleotides (n = 30) or hyaluronan (n = 30); patients received five weekly intra-articular knee injections and the follow-up period was 3 months after the end of treatment. Primary endpoint was to determine polynucleotides (PN) efficacy in reducing knee pain at the end of the study, over baseline value and over standard hyaluronan viscosupplementation (HA). Pain levels were measured using a 0-10 cm Visual Analogue Scale (VAS). Secondary endpoints included Knee Osteoarthritis Outcome Score (KOOS), NSAIDs consumption, crackling during movement and articular mobility limitation. The mean global VAS pain decreased from 5.7 + or - 1.9 cm (T0) to 1.9 + or - 1.5 cm (T16) in polynucleotide group and from 4.9 + or - 2.0 cm (T0) to 2.1 + or - 1.4 cm (T16) in hyaluronan group. The reduction in pain was statistically significant for both groups. KOOS increases from baseline values were statistically significant in both groups. No significant adverse events were reported. These findings suggest that intra-articular polynucleotides can be a valid alternative to traditional hyaluronan supplementation for the treatment of knee osteoarthritis.

Efficacy of intra-articular polynucleotides associated with hyaluronic acid vs hyaluronic acid alone in the treatment of knee osteoarthritis: A systematic review and meta-analysis of randomized clinical trial.

BACKGROUND: The reduced range of motion and pain are the most characteristic clinical features of osteoarthritis (OA). Hyaluronic acid (HA), which is one of the infiltrative therapies for OA treatment, and polynucleotides (PNs), which is a DNA-derived macromolecule favored cell growth and collagen production, are an ongoing debate in clinical effectiveness. METHODS: We plan to perform a systematic review and meta-analysis of randomized clinical trial to evaluate efficacy of intra-articular polynucleotides associated with hyaluronic acid versus hyaluronic acid alone in the treatment of knee osteoarthritis. We will search PubMed, EMBASE, Cochrane Library using a comprehensive strategy. The related conference proceedings and reference lists of the included studies will also be checked to identify additional studies. Two reviewers will screen retrieved records, extract information and assess the risk of bias independently. Stata v15.1 software will be used to conduct data synthesis. RESULTS: This study will be submitted to a peer-reviewed journal for publication. CONCLUSION: We hope it will provide a relatively comprehensive reference for clinical practice and future relevant clinical trials. ETHICS AND DISSEMINATION: Ethics approval and patient consent are not required, as this study is a systematic review and meta-analysis. PROSPERO REGISTRATION NUMBER: CRD42020167678.

Amphipathic DNA polymers exhibit antiviral activity against systemic murine Cytomegalovirus infection.

BACKGROUND: Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated. RESULTS: In vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 microM and 0.16 microM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 microM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers. CONCLUSION: These studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.

Antiviral activity of polyribocytidylic acid in cells primed with polyriboinosinic acid.

Separate administration of polyribocytidylic acid [poly(rC)] and polyriboinosinic acid [poly(rI)] to cell cultures in vitro resulted in an antiviral activity identical to or greater than that resulting from addition of the poly(rI) * poly(rC) complex. Priming of cells with poly(rI), followed by treatment with poly(rC), gave a consistently greater antiviral activity than poly(rI) * poly(rC) itself. This priming effect was obtained in several cell cultures challenged with different viruses. In vivo, the antiviral activity of poly(rI) * poly(rC) was only partially restored if poly(rI) and poly(rC) were injected separately; prior injection of poly(rI) proved superior in restoring this antiviral activity as compared to prior injection of poly(rC).

Strategies in the design of endosomolytic agents for facilitating endosomal escape in nanoparticles.

Nanoparticles (NPs) are one of the leading and promising technologies for gene and drug delivery. However, despite continuous advancements in the delivery of NPs, endosomal escape remains a major issue and a matter of grave concern for developing an efficient and targeted delivery system for therapeutic applications. Most of NPs generally follow endocytic pathway for internalization into the cells. Following the internalization process, NPs must escape into the cell cytoplasm for evading degradation by hydrolytic enzymes present in the lysosomes. Various types of lipids have been used in the past viz. fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), pH-sensitive lipids, cationic lipid and multiple charges containing lipid to escape from endosomes. Recently, several novel polymers, pH-sensitive peptides, proteins and many others endosomolytic agents have been identified and developed for incorporating into gene and drug delivery system to facilitate endosomal escape. In this review, endosomal escape mechanisms of different types of NPs have been discussed in detail and compared with endosomal escape mechanisms of viruses and other synthetic gene delivery systems to escape from endosomes. Also, the designing of endosomolytic agents to facilitate endosomal escape based on different approaches and strategies is explored. Moreover, this review article highlights the recent advancements in the development of NPs equipped with endosomolytic agents including its future directions and applications in the field of nanomedicine.

Effect of multiple intra-articular injections of polynucleotides on treatment of intractable knee osteoarthritis: A case report.

RATIONALE: Knee osteoarthritis (KOA) is a chronic joint degenerative disease. Intra-articular injection (IAI) of hyaluronic acid (HA) is widely used to treat KOA. However, some HA injections have no effect at all. Polynucleotides (PN) are recently noted as a valid substitute for HA. PATIENT CONCERNS: A 61-year-old female was admitted to the pain center with symptoms of pain over the knee and warmth feeling with stiffness in the left knee. The patient reported chronic severe pain in the left knee area despite 6 times IAI of HA. She had past medical history of breast cancer and thyroid cancer. DIAGNOSES: She was diagnosed as having KOA. INTERVENTIONS: Ultrasound-guided IAI of PN was carried out 3 times in 3 weeks. OUTCOMES: She was followed-up for more than 5 months with good improvement in intractable knee pain without any adverse event. LESSONS: IAI of PN is an efficient therapeutic option for KOA treatment if HA injection is unsuccessful.

Synthetic nanocarriers for the delivery of polynucleotides to the eye.

This review is a comprehensive analysis of the progress made so far on the delivery of polynucleotide-based therapeutics to the eye, using synthetic nanocarriers. Attention has been addressed to the capacity of different nanocarriers for the specific delivery of polynucleotides to both, the anterior and posterior segments of the eye, with emphasis on their ability to (i) improve the transport of polynucleotides across the different eye barriers; (ii) promote their intracellular penetration into the target cells; (iii) protect them against degradation and, (iv) deliver them in a long-term fashion way. Overall, the conclusion is that despite the advantages that nanotechnology may offer to the area of ocular polynucleotide-based therapies (especially AS-ODN and siRNA delivery), the knowledge disclosed so far is still limited. This fact underlines the necessity of more fundamental and product-oriented research for making the way of the said nanotherapies towards clinical translation.

Assessment of outer membrane vesicles of periodontopathic bacterium Porphyromonas gingivalis as possible mucosal immunogen.

Periodontitis is the most prevalent infectious disease and related to oral and systemic health, therefore novel prophylaxis to prevent the disease is highly desirable. Here, we assessed the outer membrane vesicles (OMVs) of a keystone periodontal pathogen, Porphyromonas gingivalis, as a candidate mucosal immunogen and adjuvant for a periodontitis vaccine. The structural and functional stability of OMVs, demonstrated by proteinase K resistance and ability to withstand long-term storage, are considered advantageous for carrying the OMV components into the host immune system. Intranasal vaccination of OMVs in mice elicited production of P. gingivalis-specific antibodies in blood and saliva by OMVs in a dose-dependent manner, which was dramatically enhanced by addition of a TLR3 agonist, Poly(I:C). Serum samples from mice immunized with OMVs plus Poly(I:C) adjuvant [OMV+Poly(I:C)] showed significant inhibition of gingipain proteolytic activity of not only the vaccine strain, but also heterologous strains. The viability of P. gingivalis was also decreased by preincubation with OMV+Poly(I:C)-immunized sera, while the killing effect was partially blocked by heat-inactivation of the sera. Saliva samples from mice immunized with OMV+Poly(I:C) enhanced bacterial agglutination of both the vaccine and heterologous strains. In an oral infection mouse model, the numbers of P. gingivalis in the oral cavity were significantly decreased in mice intranasally immunized with OMV+Poly(I:C) as compared to mock (only Poly[I:C])-immunized mice. The high levels of serum IgG (including IgG1 and IgG2a) and salivary S-IgA were elicited in mice intranasally immunized with OMV+Poly(I:C), which were maintained for at least 28 and 18weeks, respectively, after immunization. An experiment examining the accumulation of OMVs after intranasal immunization in proximal organs and an intracerebral injection experiment confirmed the safety of OMVs. Based on our results, we propose that intranasal immunization with OMV+Poly(I:C) is a feasible vaccine strategy in the context of bacterial clearance and safety.

Application of Magnesium Pyrophosphate-Based Sponge-Like Microparticles to Enhance the Delivery Efficiency and Adjuvant Effects of Polyriboinosinic-Polyribocytidylic Acid in Immune Cells.

The magnesium pyrophosphate particle (MgPP) is a unique and safe carrier that is prepared by simply mixing magnesium chloride and sodium pyrophosphate. In this study, we investigated whether MgPP can be used to deliver nucleic acid-based adjuvants to immune cells. Polyriboinosinic-polyribocytidylic acid (polyI:C), a ligand for toll-like receptor 3, was selected as a model nucleic acid-based adjuvant. PolyI:C-loaded MgPP (polyI:C-MgPP) was prepared by adding polyI:C during the MgPP preparation process. Efficient loading of polyI:C into MgPP was confirmed by measuring the absorbance at 260 nm after disruption of polyI:C-MgPP by ethylenediaminetetraacetic acid. Scanning electron microscopy revealed that both MgPP and polyI:C-MgPP had a unique sponge-like shape with a diameter of approximately 1 µm. PolyI:C-MgPP was more efficiently taken up by toll-like receptor 3-positive RAW264.7 cells than naked polyI:C, and its uptake stimulated increased tumor necrosis factor-α production. When the presentation of ovalbumin (OVA), a model antigen, was evaluated after the addition of OVA along with naked polyI:C or polyI:C-MgPP to mouse dendritic DC2.4 cells, polyI:C-MgPP substantially increased OVA presentation. These results indicate that MgPP is a useful delivery vehicle for polyI:C and that polyI:C-MgPP is an effective immune cell adjuvant.

Polynucleotide immunization of nonhuman primates against carcinoembryonic antigen.

In preparation for a Phase I trial of DNA immunization against carcinoembryonic antigen (CEA) in patients with colorectal carcinoma, we have produced a single plasmid DNA encoding CEA and hepatitis B surface antigen (HBsAg) under transcriptional regulatory control of two separate cytomegalovirus promoters within separate eukaryotic expression cassettes, designated pCEA/HBsAg. Hepatitis B surface antigen was included to provide an internal positive control for the efficacy of this immunization strategy without regard to the issue of breaking tolerance to a self-antigen. In the present work, we sought to examine the immunogenicity of this plasmid in a nonhuman primate model with close phylogenetic relationship to humans. Groups of pig-tailed macaques were immunized with pCEA/ HBsAg by i.m. injection or particle bombardment of the skin according to a dose and schedule thought to be optimal for the respective technique of DNA immunization. Both administration techniques produced humoral and lympho-proliferative responses of comparable magnitude. However, delayed type hypersensitivity to CEA and CEA-specific interleukin-2 release were observed only in the i.m. group, suggesting a qualitative difference in the character of the immune response elicited by the two techniques of DNA immunization. The antibody responses to CEA and HBsAg were surprisingly persistent in that all immunized animals maintained moderate antibody titers against both antigens for more than 15 months after the last boost. No toxicity was observed during 2 years of follow-up, including no measurable levels of anti-DNA antibody. This antitumor immunization strategy is presently being examined in patients with metastatic colorectal carcinoma using pCEA/HBsAg administered by i.m. injection.

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

Atelocollagen for protein and gene delivery.

Recent progress in recombinant gene technology and cell culture technology has made it possible to use protein and polynucleotides as effective drugs. However, because of their short half-lives in the body and the necessity of delivering to target site, those substances do not always exhibit good potency as expected. Therefore, delivery systems of such drugs are important research subjects in the field of pharmacology, and to prolong the effect of these drugs, many studies are being conducted to control the release of proteins and polynucleotides from various carrier materials. Collagen is one of the most useful carrier materials for this purpose. In this article, we report on the controlled release of protein drugs using collagen, focusing on a new drug delivery system (DDS), the Minipellet, as our basic technology. Then we introduce our recent work about gene therapy using collagen-based DDS. Basic formulation study showed that collagen DDS protects DNA degradation from both chemical cleavage and enzymatic digestion. A single injection of collagen DDS containing plasmid DNA produced physiologically significant levels of gene-encoding proteins in the local site and systemic circulation of animals and resulted in prolonged biological effects. These results suggest that collagen DDS containing plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Also, variety kinds of application of collagen DDS for gene therapy using adenovirus vector, antisense DNA and DNA vaccine, will be discussed.