Scalable Imprinting of Shape-Specific Polymeric Nanocarriers Using a Release Layer of Switchable Water Solubility

There is increasing interest in fabricating shape-specific polymeric nano- and microparticles for efficient delivery of drugs and imaging agents. The size and shape of these particles could significantly influence their transport properties and play an important role in <i>in vivo</i> biodistribution, targeting, and cellular uptake. Nanoimprint lithography methods, such as jet-and-flash imprint lithography (J-FIL), provide versatile top-down processes to fabricate shape-specific, biocompatible nanoscale hydrogels that can deliver therapeutic and diagnostic molecules in response to disease-specific cues. However, the key challenges in top-down fabrication of such nanocarriers are scalable imprinting with biological and biocompatible materials, ease of particle-surface modification using both aqueous and organic chemistry as well as simple yet biocompatible harvesting. Here we report that a biopolymer-based sacrificial release layer in combination with improved nanocarrier-material formulation can address these challenges. The sacrificial layer improves scalability and ease of imprint-surface modification due to its switchable solubility through simple ion exchange between monovalent and divalent cations. This process enables large-scale bionanoimprinting and efficient, one-step harvesting of hydrogel nanoparticles in both water- and organic-based imprint solutions

Functionality of various LKS cells.

<p>Hematopoietic differentiation of <i>in-vitro and in-vivo</i> derived Lin-ckit+Sca-1+ cells. <b>A)</b> Lin-ckit+Sca-1+ <i>in-vitro</i> derived ESCs from Static and Dynamic (Synthecon) culture conditions, as well as <b>B)</b><i>in-vivo</i> isolated LKS cells from Bone marrow (BM) were cultured for 7 days in the presence of GM-CSF, IL-4 and IL3. Approximately 10.3% of Static-derived cells (gated on FSC-A and SSC-A) are CD11c+ (Dendritic-like cells) and about 11.5% express B220+ (B cell-like progenitors). No significant expression of CD11c or B220 in Synthecon-derived cells is observed following differentiation. As expected, Lin-ckit+Sca-1+ BM cells also differentiate into CD11c and B220+ hematopoietic progenitors.</p

Differences in LKS cell gene expression.

<p>Fold changes of lineage-specific genes differentially regulated in Lin-ckit+Sca-1+ cells derived from Static and bioreactor culture conditions (Synthecon and Spinner flask) relative to LKS cells purified from BM and FL. Expression levels (n = 3) were compared for genes encoding cell surface markers of hematopoiesis (<b>A, B</b>), and blood cell markers (erythropoiesis) (<b>C,D</b>), the HSC “fingerprint” genes of Chambers et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051944#pone.0051944-Chambers1" target="_blank">[18]</a> (<b>E,F</b>) and critical hematopoietic transcription factors (<b>G,H</b>). The expression levels of hematopoietic markers (<b>A,B</b>) and blood cell markers (erythropoiesis) (<b>C,D</b>) were primarily down-regulated under ESC conditions as compared to BM and FL. When compared to BM and FL, the majority of the HSC fingerprint (<b>E,F</b>) and HSC transcription factor gene expression (<b>G,H</b>) was also down-regulated. (*) represent gene- expression values of statistical significance (p<0.05).</p

Global gene expression profile of BM- and FL-isolated as well as in vitro generated Lin-ckit+Sca-1+ progenitor cells.

<p><b>A)</b> Sample Hierarchical clustering represents the conditions that cluster together based on differentially expressed genes (DEG) across all samples (mean of gene expression values for each group, One way Anova, p<0.05). <b>B)</b> Gene Hierarchical clustering of 5-fold DEG across native (BM+FL),Static and Dynamic (Spinner+Synthecon) culture conditions. Gene Hierarchical clustering of 5-fold DEG up-regulated (<b>C</b>) or down-regulated (<b>D</b>) in Static and Dynamic (Spinner and Synthecon) <i>in vitro</i> culture conditions compared to native isolated LKS cells (BM+FL). The genes used in these heat maps are listed within Supplementary material.</p

Identification and isolation of ES cells and BM- and FL-derived Lin-ckit+Sca-1+ cells.

<p>(<b>A</b>) Lin-ckit+Sca-1+ cells were generated from ES cells differentiated under Static and Dynamic culture conditions (Synthecon and Spinner flask) for 7 days and (<b>B</b>) from BM and FL. Mouse embryonic stem cells were differentiated under Static and Dynamic culture conditions (Spinner flask and Synthecon) for 7 days. Cell suspensions from day7 EBs were stained with ckit and Sca-1 antibodies to isolate the Lin-ckit+Sca-1+ population. As explained in Materials and Methods, the Lineage negative cocktail was not included for sorting because we did not observe lineage positive cells at day 7 of differentiation for any ESC culture condition (left panel). Similarly, lineage negative cells from BM and FL cell suspensions were stained with ckit and Sca-1 antibodies to isolate by cell sorting the ckit+Sca-1+ Lineage negative population (LKS). Staining with relevant isotype controls was included in each sample to determine the right ckit+Sca-1+ population.</p