(I-332) Nanoparticle targeting of mechanically modulated glycocalyx

Introduction:
The extracellular environment of the lungs often plays an important role in disease progression.1 Specifically, in cancer and fibrosis, there is an increase in the stiffness of the lung alveolar interstitium – the basement membrane on which the epithelium grows.2 Stiffness changes can alter the behavior of alveolar epithelial cells,3,4 especially when considered in context of 3-dimensional (3D) stretching that is essential to alveolar function. Despite the importance of mechanical environment in guiding cell function and disease etiology, nanotherapeutics are not formulated to leverage important environmental cues. Here, a biomimetic platform imitating active forces and stiffness in a fibrotic tumor microenvironment was developed to culture alveolar epithelial cells to identify nanoparticle formulations that leveraged environmentally regulated changes in the epithelial glycocalyx, a sugar coating on cellular surface. Cells cultured in these platforms showed that a complex combination of stiffness and dynamic forces altered the expression of critical glycocalyx proteoglycans. Based on these changes, liposomal nanoparticles (LNP’s) were synthesized with peptides that target the cellular glycocalyx. Importantly, uptake of LNPs increased in cells cultured on stiffer interstitium with chondroitin sulfate A targeting when dynamic stretch was applied. In contrast, heparan sulfate targeting LNPs showed better cellular uptake in a static stiff environment. These results stand in stark contrast to uptake observed in conventional screening models consisting of tissue culture plastic where LNPs that do not target the glycocalyx had higher uptake. These results demonstrate the fundamental role of mechanical stretch and stiffness in the study of nanotherapeutic design for cancer treatment.


Materials and Methods:
A cell culture platform mimicking the alveolar interstitium was prepared. A thin polydimethylsiloxane (PDMS) membrane with different stiffness was prepared by spin coating. A nanofibrous membrane mimicking epithelium basement structure was prepared by electrospinning PCL in 1,1,1,3,3,3-hexafluoro-2-propanol solution. The remaining sections were prepared by casting PDMS resin and curing agent on 3D printed molds which were combined after curing. Alveolar epithelial cells (A549) were cultured in these platforms with and without air infusion at a cyclic rate with 15% linear strain at 0.2 Hz frequency (like breathing movement). The deformation was characterized through curvilinear profiling and linear strain calculation. Cells cultured at 37 °C, 5% CO2 with Dulbecco's Modified Eagle Medium (DMEM), L-glutamine, 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin. Glycocalyx expression of cells were observed by extracting RNA followed by cDNA preparation and qPCR. Liposomal nanoparticles were synthesized with 58% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 42% Cholesterol and Vybrant Dil/DiO fluorophore utilizing thin film hydration. Peptide conjugates that selectively target lung cancer were prepared by reacting cysteine terminated peptides with twice the molar amount of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol) (DSPE-PEG20kD) maleimide in anhydrous DMSO with triethylamine overnight at 4 ℃. They were purified by high-performance liquid chromatography and characterized by gel electrophoresis. Overnight incubation of liposomes with peptide conjugates completed the synthesis of peptide decorated liposomes which was confirmed by dynamic light scattering. Liposomes were incubated with cells for 4 hours in DMEM at 0.15nM concentration. Flow cytometry was performed to quantify accumulation of peptide decorated LNPs inside cells.  


Results, Conclusions, and Discussions:
The biomimetic platform effectively applies mechanical forces present in diseased lungs. Cells adhering to nanofibrous membrane were cultured on PDMS membranes with stiffness mimicking normal (1-5 kPa) and fibrotic (20-100 kPa) conditions. Air infusion applied 3-D stretch to membranes providing mechanical stimulation to cells (Figure 1a).

qPCR of heparan sulfate proteoglycans (SDC1, SDC2, SDC4) and chondroitin sulfate proteoglycans (VCAN, DCN) showed upregulation of SDC1, VCAN and downregulation of DCN in biomimetic platform. SDC2 was upregulated in dynamic stiff, SDC4 in dynamic soft and VCAN, DCN in static soft conditions compared to TCP. Thus, a complex combination of stiffness and dynamics drives glycoprotein expression (Figure 1b).

LNPs were prepared with chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and heparan sulfate (HS) recognizing peptide conjugates attached to their surface (Figure 1c). LNPs incubated with cells in biomimetic substrates resulted in enhanced uptake depending on the peptide. CSA recognition peptides promoted uptake into cells grown on stiff environment with dynamic stretch and HS recognition peptides facilitated uptake into cells on stiff membranes without stretch. In contrast, cells grown on TCP selectively took up unmodified liposomes (Figure 1d).

In conclusion, nanoparticle designs targeting glycocalyx properties driven by the tissue environment have the potential to improve therapeutic efficiency for lung cancer. However, higher proportions of glycocalyx proteoglycan does not always enhance uptake but acts as barriers. Screening approaches considering biomimetic conditions can bridge the gap between in vivo and in vitro studies, and exploring multiple glycocalyx proteoglycan targeting combinations can further enhance therapeutic efficiency.



Acknowledgements (Optional):

References (Optional):
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(2) Kuhn, C.; McDonald, J. A. The Roles of the Myofibroblast in Idiopathic Pulmonary Fibrosis. Ultrastructural and Immunohistochemical Features of Sites of Active Extracellular Matrix Synthesis. Am. J. Pathol. 1991, 138 (5), 1257–1265.

(3) Huang, C.; Butler, P. J.; Tong, S.; Muddana, H. S.; Bao, G.; Zhang, S. Substrate Stiffness Regulates Cellular Uptake of Nanoparticles. Nano Lett. 2013, 13 (4), 1611–1615. https://doi.org/10.1021/nl400033h.

(4) Mahmoud, M.; Cancel, L.; Tarbell, J. M. Matrix Stiffness Affects Glycocalyx Expression in Cultured Endothelial Cells. Front. Cell Dev. Biol. 2021, 9, 731666. https://doi.org/10.3389/fcell.2021.731666.