Investigating Cell Uptake of Guanidinium-Rich RAFT Polymers: Impact of Comonomer and Monomer Distribution

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEA^diBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited

Aggregation and Chondrogenesis of sfMPCs.

<p>Normal and OA CD90+ sfMPCs were induced to differentiate without prior micro-mass aggregation. By day 14 normal sfMPCs expressed Sox9, Collagen2 and Aggrecan mRNA (A,D) while OA sfMPCs expressed severely reduced levels (A,D). Furthermore, normal sfMPCs generated a tissue-like structure that stained positive for Alcian Blue (B,G), whereas OA sfMPCs did not (C,F) and remained as a monolayer. Scale bars represent 200 µM.</p

Primary antibodies used for analyses.

<p>Primary antibodies used for analyses.</p

Staining and TEM of Derived Tissue from Normal sfMPCs.

<p>The resultant tissue-like masses were stained with antibodies to Collagen 2 (A) and Aggrecan (C), nuclei were stained with TOTO3 (B, D). Cells were found in closer association to each other (E), with higher magnification showing production of collagen fibres (F). Cells were also found in proximity with the produced collagen ECM (E, F). Scale bars represent 200 µM.</p

qRT-PCR probes used for analysis.

<p>qRT-PCR probes used for analysis.</p

Multi-potent differentiation of CD90+/− sfMPCs.

<p>Normal and OA CD90+ or CD90− sfMPCs were induced to differentiate into osteoblasts or adipocytes and stained with Akaline phosphatase (A) to indentify osteoblasts, or Oil Red-O (B) to identify lipid-containing cells (adipocytes). qRT-PCR was used to determine the relative level of adipocyte specific genes (Adiponectin, PPAR-gamma, [C]) and osteoblastic specific genes (Osteonectin, Sp7/Osterix [D]). * = p>0.05.</p

FACS characterization of human sfMPCs.

<p>Normal and OA CD90+ and CD90− subpopulations were assayed for expression of CD105, CD90, CD73, CD45 and CD11b prior to chondrogenic differentiation.</p

Micro-mass differentiation of sfMPCs.

<p>Normal (human N = 5 A–D & canine N = 2 H–K) and OA (human N = 5 E–G & canine N = 2 L–N) derived sfMPCs where aggregated using the micro-mass technique and induced to differentiate over a 14 day period with media supplements. qRT-PCR results demonstrated that Sox9, Collagen 2 and Aggrecan are significantly elevated at days 3, 5, 8 and 14 of differentiation (A,E,H,L), furthermore, at day 14 Collagen 2 protein is expressed in the cell cultures (B,F,I,M). Secondary controls (C,J) demonstrate minimal non-specific staining. Scale bars represent 50 µM. * = p>0.05.</p

Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

<div><p>Background</p><p>The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.</p><p>Methods</p><p>Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist's report.</p><p>Results</p><p>15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.</p><p>Conclusions</p><p>This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.</p></div

Consultant's Opinion of 263 Patients in the ARC.

<p>*Other includes gout, tendonitis, bursitis, patellofemoral syndrome, palindromic rheumatism, spondyloarthropathy, psoriatic arthritis, unspecified polyarthropathy.</p