In Vivo GFP Knockdown by Cationic Nanogel-siRNA Polyplexes.

RNA interference (RNAi) is a powerful tool to treat diseases and elucidate target gene function. Prior to clinical implementation, however, challenges including the safe, efficient and targeted delivery of siRNA must be addressed. Here, we report cationic nanogel nanostructured polymers (NSPs) prepared by atom transfer radical polymerization (ATRP) for in vitro and in vivo siRNA delivery in mammalian models. Outcomes from siRNA protection studies suggested that nanogel NSPs reduce enzymatic degradation of siRNA within polyplexes. Further, the methylation of siRNA may enhance nuclease resistance without compromising gene knockdown potency. NSP-mediated RNAi treatments against Gapdh significantly reduced GAPDH enzyme activity in mammalian cell culture models supplemented with 10% serum. Moreover, nanogel NSP-mediated siRNA delivery significantly inhibited in vivo GFP expression in a mouse model. GFP knockdown was siRNA sequence-dependent and facilitated by nanogel NSP carriers. Continued testing of NSP/siRNA compositions in disease models may produce important new therapeutic options for patient care.

Nanogel-Mediated RNAi Against Runx2 and Osx Inhibits Osteogenic Differentiation in Constitutively Active BMPR1A Osteoblasts.

Trauma-induced heterotopic ossification (HO) and fibrodysplasia ossificans progressiva (FOP) are acquired and genetic variants of pathological bone formation occurring in soft tissues. Conventional treatment modalities target the inflammatory processes preceding bone formation. We investigated the development of a prophylaxis for heterotopic bone formation by addressing the biological basis for HO - dysregulation in the bone morphogenetic protein (BMP) signaling pathway. We previously reported the synthesis of cationic nanogel nanostructured polymers (NSPs) for efficient delivery of short interfering ribonucleic acids (siRNAs) and targeted gene silencing. Results suggested that nanogel:siRNA weight ratios of 1:1 and 5:1 silenced Runx2 and Osx gene expression in primary mouse osteoblasts with a constitutively active (ca) BMP Receptor 1A (BMPR1A) by the Q233D mutation. Repeated RNAi treatments over 14 days significantly inhibited alkaline phosphatase activity in caBMPR1A osteoblasts. Hydroxyapatite (HA) deposition was diminished over 28 days in culture, though complete suppression of HA deposition was not achieved. Outcome data suggested minimal cytotoxicity of nanogel-based RNAi therapeutics, and the multistage disruption of BMP-induced bone formation processes. This RNAi based approach to impeding osteoblastic differentiation and subsequent bone formation may form the basis of a clinical therapy for heterotopic bone formation.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells.

BACKGROUND: Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. QUESTIONS/PURPOSES: We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. METHODS: Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay). RESULTS: Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures. CONCLUSIONS: Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis. CLINICAL RELEVANCE: Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.

Synthesis of poly(meth)acrylates with thioether and tertiary sulfonium groups by ARGET ATRP and their use as siRNA delivery agents.

The field of RNA interference depends on the development of safe and efficient carriers for short interfering ribonucleic acid (siRNA) delivery. Conventional cationic monomers for siRNA delivery have utilized the nitrogen heteroatom to produce cationic charges. Here, we polymerized cationic sulfonium (meth)acrylate by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) to form polymers with narrow molecular weight distributions for siRNA delivery. The tertiary sulfonium species was stable toward dealkylation in water but less stable in the polar aprotic solvent dimethyl sulfoxide. Block copolymers poly(ethylene oxide) with poly(meth)acrylate containing sulfonium moieties were prepared as an siRNA delivery platform. Results suggested block copolymers were biocompatible up to 50 µg/mL in vitro and formed polyplexes with siRNA. Additionally, block copolymers protected siRNAs against endonuclease digestion and facilitated knockdown of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA expression in murine calvarial preosteoblasts. The versatility, biocompatibility, and cationic nature of these tertiary sulfonium groups are expected to find widespread biological applications.

Cationic nanostructured polymers for siRNA delivery in murine calvarial pre-osteoblasts.

The endogenous RNA interference (RNAi) pathway enables control of pathologies caused by the dysregulation of proteins. Several biological molecules are active in RNAi including short interfering ribonucleic acid (siRNA). The effective utilization of siRNA as a therapeutic agent has been marked with distinct challenges, namely in intracellular delivery and achieving a sufficient dosage to affect protein expression. A delivery strategy we have developed to improve safety and efficacy of siRNA includes complexing siRNA with nanostructured polymers delivery systems (NSPs). These NSPs are synthesized via atom transfer radical polymerization (ATRP) and combine several important advances in polymer architecture for siRNA delivery. This includes shielding the cationic charge of the NSP with a poly(ethylene glycol) (PEG) shell to promote cell viability in MC3T3-E1.4 pre-osteoblasts, and minimize the inflammatory response in a C57BL/6 mouse model. In our gene knockdown experiments targeting glyceraldehyde 3-phosphate dehydrogenase Gapdh expression, star polymer and nanogel polyplexes suppressed Gapdh mRNA to levels comparable to cells treated with Lipofectamine RNAiMAX lipoplexes.

Bone tissue engineering: state of the union.

The quest to surpass the clinical efficacy of the allogeneic bone graft has had limited success, an outcome that is symbolic of tissue engineering as a whole. In this 'State of the Union'-type review, we highlight recent advances in the design of bone regenerative therapeutics using the primary elements of stem cells, growth factors and scaffolds, and identify major obstacles in their paths to the clinic. We underscore the need for rigorous performance criteria in the design of holistic tissue regenerative therapeutics, and an increased emphasis on the product production, storage and handling issues that will ultimately influence clinical success.

The delivery and evaluation of RNAi therapeutics for heterotopic ossification pathologies.

RNA interference (RNAi) is a powerful tool being used to develop therapies for pathologies caused by gene overexpression. Heterotopic ossification pathologies such as trauma-induced heterotopic ossification and fibrodysplasia ossificans progressiva may be treatable with an RNAi approach. However, there is a lack of consensus in literature regarding the delivery conditions and evaluation of RNAi therapeutics in these disease models. Here, we describe in vitro protocols for the delivery of polymer-based RNAi therapeutics as well as a streamlined strategy for the assessment of osteoblast lineage progression due to dysregulated bone morphogenetic protein signaling. This strategy focuses on the quantification of early-stage osteoblast transcription factors RUNX2 and OSX, followed by the measurement of alkaline phosphatase activity and late-stage matrix deposition.

Preparation of cationic nanogels for nucleic acid delivery.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery.

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 µg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.