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h-Amino ester adjuvanting of plasmid DNA augments peak CD8 + cellular immune responses. (A) Mice (n = 10) were immunized with 50 Ag of pGWiz-LacZ plasmid formulated with 100 Ag of the indicated polymers. Spleens were harvested 12 days following immunization and cultured with a dominant h-galactosidase-specific peptide epitope for 18 h to determine the anti-LacZ responses. (B) Mice (n = 7) were immunized with 50 Ag of pVRC- gp120 formulated with 100 Ag of the indicated polymers. Peak (day 12) and memory (day 28) cellular immune responses were measured by p18-tetramer binding to peripheral blood CD8 + lymphocytes. Asterisk denotes P b 0.05 by a Mann–Whitney test comparing the immune response to that elicited by plasmid DNA alone.
Source publication
Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candi...
Contexts in source publication
Context 1
... alone or formulated with 100 Ag of CRL1005, U28, or JJ28. We chose these two h-amino ester polymers because they had induced the highest levels of transgene activity in vivo. We isolated splenocytes 12 days following inoculation and analyzed them for a dominant CTL-specific H-2 L d - restricted h-galactosidase epitope by Elispot. As shown in Fig. 4A, the highest frequency anti-h-galactosidase responses were observed in mice inoculated with plasmid DNA adjuvanted with the two h-amino ester ...
Context 2
... and H-2 D d /p18 tetramer (Fig. 4B). As observed in the study using the h-galactosidase immunogen, higher frequency peak immune responses were observed in the groups of mice that received h-amino esters, with the exception of D94, than were seen in mice receiving plasmid DNA alone or plasmid DNA formulated with CRL1005. The immune responses elicited by the plasmid DNA ...
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Citations
... DNA delivery and tissue engineering [131][132][133][134][135][136][137][138] Polyhydroxyalkanoates (PHAs) ...
... Gene delivery, drug delivery, and tissue engineering [127,128] Polyalkylcyanoacrylates Drug delivery and oxygen carriers for blood substitutes [129,130] Polyamides DNA delivery and tissue engineering [131][132][133][134][135][136][137][138] Polyhydroxyalkanoates (PHAs) ...
Bioresorbable nanomembranes (NMs) and nanoparticles (NPs) are powerful polymeric materials playing an important role in biomedicine, as they can effectively reduce infections and inflammatory clinical patient conditions due to their high biocompatibility, ability to physically interact with biomolecules, large surface area, and low toxicity. In this review, the most common bioabsorbable materials such as those belonging to natural polymers and proteins for the manufacture of NMs and NPs are reviewed. In addition to biocompatibility and bioresorption, current methodology on surface functionalization is also revisited and the most recent applications are highlighted. Considering the most recent use in the field of biosensors, tethered lipid bilayers, drug delivery, wound dressing, skin regeneration, targeted chemotherapy and imaging/diagnostics, functionalized NMs and NPs have become one of the main pillars of modern biomedical applications.
... tential as DNA vaccines against HIV (Greenland et al., 2005). The in vivo immunogenicity of the PBAE polymer encapsulating plasmid DNA was compared with poloxamer CRL1005, and several polymer formulations showed 4-to 10-fold higher activity in expression of β-galactosidase as model protein after intramuscular injection of 50 μg of DNA. ...
Translation of nanoparticle-based therapeutics from preclinical to clinical stage has seen slow progress compared with their small molecules counterparts primarily due to lack of suitable technologies that can map the high-throughput combinatorial chemistry-based approach of molecule design. Our improved understanding of materials chemistry and process control parameters, however, has recently led to a surge in the application of combinatorial design to develop a library of materials for synthesizing nanoparticles. Combinatorial chemistry of materials uses a common backbone of starting materials with the aim of developing different ". building blocks" that possess the desired individual property. These individual blocks can have properties such as enhanced encapsulation of the therapeutic moiety, protection against biological barriers, improved in vivo residence time, or diagnostic component to obtain a theranostic system. These individual blocks can be ". mixed and matched" to obtain a library of nanoparticle formulations, which have a combination of properties owing to the individual components. Such library of formulations can be screened for desired physical and biochemical characteristics and can be further challenged with in vitro and in vivo tests for candidate selection. This chapter will specifically focus on the use of polymeric backbone to develop a library of novel materials and their subsequent biomedical applications. The illustrative examples from preclinical studies cement the promise of the field and highlight the strength of this approach toward developing next-generation nanotherapeutics.
... Non-PEGylated PBAE-CN was formulated as previously reported [23,54,57,68,69,72,75]. PBAE-BPN were formulated using a protocol we have recently established [23]. ...
... We then performed qPCR to compare the osteogenic marker expression levels between the engineered and wild-type hADMSCs. Biodegradable, biocom- 47,48 was used for pTAZ plasmid transfection into hADMSCs. Particularly, C32−122, PBAE modified with 122 amine at both ends of the polymer backbone ( Figure 3A), has been found to be highly efficient in delivering plasmid genes into various types of cells including MSCs. ...
The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.
... Blending a pH sensitive, cationic polymer PBAE with PLGA for microparticle fabrication using double emulsion/solvent evaporation leads to increased pDNA encapsulation efficiency and enhanced intracellular delivery when compared with PLGA alone [20]. Due to their low toxicity, biodegradability, and capacity to electrostatically interact with anionic biomolecules such as pDNA, PBAE-mediated pDNA delivery complexes have been widely explored as a nonviral gene delivery vector [128]. One application using PBAE nanocomplex is VEGF transfection in stem cells [129] and HUVECs [130] for the treatment of ischemic diseases. ...
Polycations are useful delivery vehicles for nucleic acids and proteins. Physicochemical properties, safety, and cost are important design parameters for polycation-enabled controlled release methods. Improvements in the design and biocompatibility of synthetic polycations and complexes thereof are necessary for clinical applications. This review focuses on breakthroughs in the development of biocompatible polycations and their biomedical applications in the past 10 years. First, we summarize current strategies to develop naturally derived and synthetic polycations and describe the most commonly used polycations. Second, we discuss polycation-mediated non-viral gene delivery systems used for tissue engineering and regenerative medicine. Third, we review the development of polycation-mediated self-assembled systems for the delivery of heparin-binding proteins, with an emphasis on translational potential. Finally, we introduce platforms for fabricating polycation-based complexes, including layer-by-layer assembly, polymeric vesicles, polycation-containing microspheres, and approaches to improve the functionality of delivery complexes. With improvements in polycation design, safety, and efficacy, polycation-based controlled delivery is expected to contribute significantly to tissue repair and regeneration applications.
... Among several promising polymers, poly (β-amino esters) (PBAEs) provide a library of nontoxic, biodegradable polymers for the compaction of nucleic acids. Numerous high-throughput in vitro screening studies of PBAEbased gene vector libraries have shown efficient gene transfer in a cell-specific manner (5)(6)(7)(8)(9)(10)(11)(12)(13). However, in vitro behavior of gene vectors does not usually predict in vivo performance, largely due to harsh physiological environments and a variety of extracellular barriers (14)(15)(16). ...
... We established a library of PBAE polymers that consists of the polymer variants previously shown to provide efficient in vitro gene transfer (Fig. S1) (7)(8)(9)(10)(11)(12)(13). PBAE polymers were synthesized by a two-step Michael addition, as previously reported (Fig. 1) (6-13, 34). ...
Significance
Therapeutically relevant lung gene therapy is yet to be achieved. We introduce a highly translatable gene delivery platform for inhaled gene therapy based on state-of-the-art biodegradable polymers, poly(β-amino esters). The newly designed system is capable of overcoming challenging biological barriers, thereby providing robust transgene expression throughout the entire luminal surface of mouse lungs. Moreover, it provides markedly greater overall transgene expression in vivo compared with gold standard platforms, including a clinically tested system. The clinical relevance is further underscored by the excellent safety profile as well as long-term and consistent transgene expression achieved following a single and repeated administrations, respectively.
... Anderson et al. subsequently demonstrated the ability of these PBAE derivatives to be photocrosslinked to manipulate degradation behavior (55). Greenland et al. then assessed some of these PBAE candidates and showed improved in vivo transfection efficiency of plasmid DNA adjuvants for vaccine application (56). Poly[(1,5di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] showed a seven-fold increase in gene expression and a 70% enhancement in subsequent immune response. ...
With the advent of novel and personalized therapeutic approaches for cancer and inflammatory diseases, there is a growing demand for designing delivery systems that circumvent some of the limitation with the current therapeutic strategies. Nanoparticle-based delivery of drugs has provided means of overcoming some of these limitations by ensuring the drug payload is directed to the disease site and insuring reduced off-target activity. This review highlights the challenges posed by the solid tumor microenvironment and the systemic limitations for effective chemotherapy. It then assesses the basis of nanoparticle-based targeting to the tumor tissues, which helps to overcome some of the microenviron-mental and systemic limitations to therapy. We have extensively focused on some of the tumor multidrug resistance mechanisms (e.g., hypoxia and aerobic glycolysis) that contribute to the development of multidrug resistance and how targeted nano-approaches can be adopted to overcome drug resistance. Finally, we assess the combinatorial approach and how this platform has been used to develop multifunctional delivery systems for cancer therapy. The review article also focuses on inflammatory diseases, the biological therapies available for its treatment, and the concept of macrophage repolarization for the treatment of inflammatory diseases.
... Greenland et al. screened potential candidates from the PBAE library to identify polymers that show enhanced in vivo transfection efficiency of plasmid DNA adjuvants for vaccine application. 112 Among the tested polymer members, poly[(1,5-di(acryloxyethoxy)hexane)-co-(4aminobutanol) show the best activity with a seven-fold increase in gene expression and a 70% enhancement in subsequent immune response in Balb/c mice. Based on the results obtained from screening the polymer library for gene delivery, the authors could also conclude that in general, polymers with a moderate hydrophobic backbone were more effective in transfection efficiency in vivo. ...
There have been significant advances in our understanding of cancer as a disease at the molecular level. Combined with improved diagnostic systems, the concept of personalized medicine was introduced where therapy for every patient can be customized according to their disease profile. The nanotechnology approach for formulation design and the advent of drug delivery systems for small molecules and biologics has contributed to the development of personalized medicine. Despite the progress, effective management and treatment of cancer remains a clinical challenge. The majority of drug delivery vectors that have undergone clinical trials have been discontinued prematurely because of poor therapeutic outcomes, off-target effects and non-specific toxicity due to the components of the formulation itself. Therefore, there is an urgent unmet requirement for a systematic approach to design drug delivery vectors that not only deliver the cargo to the desired site of action, but are also highly biocompatible and non-toxic. The past decade has seen the evolution of a combinatorial approach to drug delivery, a concept that has been classically successful in drug discovery research. In the present review, we summarize the wet-lab and in silico strategies to designing libraries of biocompatible delivery materials using combinatorial chemistry and support this strategy with pre-clinical success stories in cancer therapy.
... To detect T and B lymphocytes responding to mycoplasma antigens, splenocytes from mice infected 2 wks were subjected to immunosorbent assays (31), as detailed in Supplemental methods. ...
Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via involvement in antigen presentation pathways. Hypothesizing that Ctsl helps to combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to airway inoculation of mycoplasma were compared in Ctsl-/- and Ctsl+/+ mice. After infection, Ctsl-/- mice had more body weight loss, greater mortality (22% vs. 0%), and heavier lungs than Ctsl+/+ mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl-/- mice than in Ctsl+/+ mice after infection for 3 d. Ctsl-/- mice had more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl+/+ mice. Compared to the conspicuous remodeling of lymphatics after infection in Ctsl+/+ mice, little lymphangiogenesis occurred in Ctsl-/- mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl+/+ mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFNgamma response to mycoplasma antigen. These findings suggest that Ctsl helps to contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and predict that therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.
... Profilometry measurements performed on PEMs constructed in parallel on Si substrates showed linear multilayer growth with increasing deposition cycles, as previously reported for (PBAE/pDNA) films ( Fig. 2b) 19,21 . Confocal imaging of microneedles coated with composite (uv-bPNMP)(PS/SPS) 20 (poly-1/Cy5-pLUC) 35 PEM films showed conformal co-localized fluorescence from SAv-labelled uv-bPNMP and Cy5-pLUC over the surface of each PLLA microneedle (Fig. 2c). (Individual uv-bPNMP and PEM films were too thin to resolve as distinct layers.) ...
... Sequential assembly of PEM films comprising layers of (poly-1/poly(I:C)) followed by layers of (poly-1/pLUC) generated microneedles coated with complete 20 films on silicon substrates as a function of the number of deposited (PBAE/pLUC) bilayers as measured by surface profilometry. Data represent the mean ± s.e.m., n = 8. c, Representative confocal images of PLLA microneedles coated with (SAv488-bPNMP)(PS/SPS) 20 (poly-1/Cy5-pLUC) 35 films (left, transverse optical sections; right, lateral sections, 100 µm z-intervals, scale bars, 200 µm; blue, Sav488-uv-bPNMP; yellow, Cy5-pLUC). d, Quantification of Cy5-pLUC and Sav488-bPNMP incorporated into (SAv488-bPNMP)(PS/SPS) 20 (poly-1/Cy5-pLUC) n films on microneedles through confocal fluorescence intensity analysis (left axis, n = 15) and measurement of total DNA recovered from dissolved films (right axis, n = 3). ...
... To test PEM film release from microneedle arrays, dried composite (SAv-labelled uv-bPNMP)(PS/SPS) 20 (poly-1/Cy5-pLUC) 35 coatings (referred to henceforth as PNMP/PEM films) on microneedles were immersed in pH 7.4 PBS for varying times in vitro and imaged by confocal microscopy to quantitate uv-bPNMP and Cy5-pLUC fluorescence remaining on the microneedle surfaces. After 15 min incubation in PBS, we observed a significant loss of both bPNMP and Cy5-pLUC fluorescence from microneedle arrays ( Supplementary Fig. S3). ...
DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These 'multilayer tattoo' DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.
