Evaluation of Histidylated Arginine-Grafted Bioreducible Polymer To Enhance Transfection Efficiency for Use as a Gene Carrier

To increase cellular uptake and endosomal escape efficiency, various methods have been studied to efficiently deliver plasmid DNA (pDNA) into the cell. Here, we designed a histidylated arginine-grafted bioreducible polymer (HABP) as a nonviral gene carrier using different ratios of histidine and arginine-grafted bioreducible poly­(cystaminebis­(acrylamide)-diaminohexane) (poly­(CBA-DAH)), known as ABP, to increase cellular uptake and endosomal escape efficiency. HABPs consist of arginine (cell penetrating functionality), histidine (endosome buffering functionality), and a disulfide bond backbone (bioreducible functionality in cytoplasm). These components result in the following: (1) polyplexes are easily taken up by cells, (2) polyplexes can easily escape from the endosome into the cytosol, and (3) pDNA can dissociate from polyplexes in reducing environments such as the cytoplasm. HABPs showed increased buffering capacity over histidine-ungrafted ABP, and HABPs formed nanosized polyplexes with pDNA. These polyplexes were about 90 nm in size and had positive charges of about of 30–40 mV. HABPs/pDNA polyplexes showed enhanced transfection efficiency and no significant cytotoxicity in comparison with polyethylenimine 25 kDa (PEI 25k), histidine-ungrafted ABP, and Lipofectamine (commercial reagent) in human cervical carcinoma (HeLa), rat cardiomyocytes (H9C2), and colon carcinoma (CT26) cells

Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens

L_1p-FH successfully attach to mSMG and are degraded over time <i>in vivo</i>.

<p>(A) Rheology measurements were performed for FH alone as well as L_1p-FH. Data represent the elasticity (G’) versus strain (%) of unmodified FH (○) and L_1p-FH (□). The <i>in vivo</i> stability of L_1p-FH was monitored using a Xenogen IVIS 100 Bioluminescent Imager at days (B) 1, (C) 3, (D) 8 and (E) 20. Radiant Efficiency: (p/sec/cm²/sr)/(μW/cm²).</p

Procedure to create wounded SMG model.

<p>(A) A skin incision of approximately 1 cm in length was made along the anterior surface of the neck, mSMG were exposed, (B) a 3-mm diameter biopsy punch was performed, surgical wounds were completed, (C) wounds were filled with or without L_1p-FH or FH and (D) the skin incision was sutured.</p

L_1p-FH applied to mSMG increased body weight.

<p>Changes in body weight (%) of FH alone (■) or L_1p-FH (▲) treated mice groups were compared with untreated mice group (●) and sham control group (○) over 20-day period. Data represent the means ± SD of n = 7 mice per condition and statistical significance was assessed by two-way ANOVA (<i>p</i> < 0.01) and Dunnett's post-hoc test for multiple comparisons to the untreated group.</p

Surgical wounds treated with L_1p-FH displayed organized mSMG.

<p>Rehydrated sections were stained with hematoxylin-eosin (A, C, E, G) or picrosirius red (B, D, F, H) stains and analyzed using a Leica DMI6000B at 10× magnifications. Shown are wounded mSMG without scaffold (A, B), wounded mSMG with FH alone (C, D), wounded mSMG with L_1p-FH (E, F), and sham control (G, H). (I) The ratio of acinar and ductal structures was analyzed using ImageJ. Red arrows indicate acinar structures and yellow arrows indicate ductal structures. Scale bars = 200 μm.</p

Composition of fibrin hydrogels.

<p>Composition of fibrin hydrogels.</p

L_1p-FH applied to mSMG improved saliva secretion over untreated and FH alone-treated mice.

<p>Mice were anesthetized and stimulated with pilocarpine at day 20. Then, saliva was collected for 5 min. Data represent the means ± SD of n = 5 mice per condition and statistical significance was assessed by one-way ANOVA (<i>p</i> < 0.01) and Dunnett's post-hoc test for multiple comparisons to the untreated group. * = significant difference from the untreated group; n.s. = no significant difference from the untreated group.</p

List of antibodies.

<p>List of antibodies.</p

L_1p-FH applied to mSMG restored saliva composition.

<p>Fifteen microgram of saliva protein from each group was fractionated by SDS-PAGE. The gel was stained with (A) 0.25% Coomassie Brilliant Blue R-250 for total proteins and (B) 0.5% Alcian Blue 8GX for mucins. (C) The mucin compositions were analysed using ImageJ. The white bar indicates MUC5B and the gray bar indicates MUC7. Statistical significance was assessed by one-way ANOVA (<i>p</i> < 0.01) and Dunnett's post-hoc test for multiple comparisons to the sham group. * = significant difference from the sham group; n.s. = no significant difference from the sham group.</p