Lili Zou

Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders.

Recent advances in peptides for enhancing transdermal macromolecular drug delivery.

Transdermal delivery of drugs, a compelling route of systemic drug delivery, provides painless, reliable, targeted, efficient and cost effective therapeutic regimen for patients. However, its use is limited by skin barrier especially the stratum corneum barrier. Moreover, transdermal delivery of macromolecules remains a challenge. Naturally, varieties of physical methods, chemical enhancers and drug carriers have been used to counteract this limitation. Recently, transdermal peptides discovered as safer, more efficient and more specific enhancers could promote the delivery of macromolecules across the skin. Herein, the underlying transdermal peptides are included. Subsequently, we have discussed typical applications and the possible mechanism of two groups of biologically inspired transdermal peptide enhancers, namely cell penetration peptides and transdermal enhanced peptides.

A New Method to Increase the Adsorption of Protein‐Bound Toxins in Artificial Liver Support Systems

In this work, a new method, called the preconcentration method (PCM), is proposed to increase the adsorption of protein-bound toxins onto adsorbents in artificial liver support systems. In the PCM, a concentrator is installed before the inlet of the adsorbent cartridge. This method is validated in an experiment using activated carbon to remove albumin-bound bilirubin, and the mechanism of the increase in adsorption is theoretically explained with breakthrough curve and equilibrium adsorption analyses. Our results show that when this PCM is used, the mass transfer rate of bilirubin from solution to activated carbon is enhanced, the adsorbed bilirubin amount per unit mass of activated carbon is greatly increased, and more albumin-bound bilirubin molecules are quickly removed from the albumin solution. When the concentration ratio (the ratio of the inlet flow rate to the outflow rate of the concentrator) is 2.59, the adsorption efficiency of activated carbon at 120 min is increased by approximately 36%. Only approximately 60 min is required for the bilirubin concentration to decrease from 19.3 to 13.0 mg/dL; however, without the PCM, nearly 180 min is needed. In addition, by adjusting the concentration ratio, the adsorption of albumin-bound bilirubin onto activated carbon can be further increased.

Unloading of cryoprotectants from cryoprotectant-loaded cells on a microfluidic platform

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (CG), the survival rate of cells (SC) and the recovery rate of cells (RC) have been studied. Under the designed conditions, CG achieved ~80% and SC reached ~90%. However, RC was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high CG and SC, but low RC. For a low glycerin concentration, CG, SC, and RC were all high. For a high cell density, CG was high, but both SC and RC were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.

Fatigue damage to pig erythrocytes during repeated swelling and shrinkage.

During the removal of cryoprotectants from cryopreserved-thawed blood with the dialysis-based or dilution-filtration method, due to the change in the extracellular osmolality, erythrocytes usually undergo repeated swelling and shrinkage. However, the erythrocyte fatigue damage induced by this repeated volume change has not yet been studied. In this work, by successively loading hypotonic and hypertonic solutions, we mimicked the repeated swelling and shrinkage of pig erythrocytes and then examined the effect of the number of cycle loops on the steady-state volume and the mortality of the pig erythrocytes. The results suggest that because of cell leakage in the swelling process, the steady-state volume of the pig erythrocytes after one cycle is smaller than the volume before the cycle, even though the cell performs a self-protective regulatory procedure. If the number of cycle loops is increased, the repeated swelling and shrinkage will cause a continuous decrease in the steady-state volume, and the ability of the pig erythrocytes to resist osmotic damage will decrease; as a result, the mortality of the pig erythrocytes increases as the number of cycle loops increases. The viability of the cells is also affected by the hypotonic and isotonic processing times: a short processing time may contribute to a decrease in the mortality of the pig erythrocytes. This work is of significance to optimizing the process of removing cryoprotectants.

A multistage-dialysis microdevice for extraction of cryoprotectants

In this study, we present a multistage-dialysis microdevice (MDM) for extraction of cryoprotectants (CPAs) from a CPA-laden cell suspension. We confirmed the functions of the key designs of the MDM using a fluorescence solution, we assessed the performance of the MDM by using the MDM to unload glycerin from glycerin-loaded swine erythrocytes, and we investigated the effects of the cell suspension flow rate, glycerin concentration, cell density, and membrane pore size on the clearance efficiency of glycerin (CG), the survival rate of cells (SC), and the recovery rate of cells (RC). Under the designed conditions, CG, SC, and RC reached ~60%, ~90%, and ~70%, respectively. In addition, a high flow rate causes high SC and RC but a low CG. For a low glycerin concentration, CG, SC, and RC are all high. If a low cell density or a large pore membrane is used, CG is high, whereas both SC and RC are low. This work provides insight into the development of microfluidic devices for the inline extraction of cryoprotectants from a small volume of cryopreserved cells prior to the use of the cells in lab-on-a-chip applications.

Cell blebbing upon addition of cryoprotectants: a self-protection mechanism.

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.

Deglycerolization of red blood cells: A new dilution-filtration system

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.

Peptide-modified vemurafenib-loaded liposomes for targeted inhibition of melanoma via the skin

Vemurafenib is a chemotherapeutic drug recently approved by the FDA to treat melanoma. Because the drug is usually delivered orally, the route of administration readily causes damage to major organs with limited antitumor efficacy and bioavailability. In this study, we developed a peptide-modified vemurafenib-loaded liposome for the targeted inhibition of subcutaneous melanoma via the skin. First, the peptide-modified vemurafenib-loaded liposomes (Vem-TD-Lip) were prepared and characterized with respect to the size, shape and charge; the loading efficiency of vemurafenib; and the stability. Then, the intracellular uptake of these liposomes, their limited cytotoxicity, the selective inhibition of melanoma cells harboring BRAF mutations, and the liposome permeation ability were confirmed through in vitro experiments. Finally, the safety and antitumor activity of Vem-TD-Lip were evaluated in vivo. The results showed that transdermal delivery of Vem-TD-Lip effectively targeted and inhibited subcutaneous melanoma in male mice, the administration of Vem-TD-Lip through skin was better than that through oral administration and intravenous injection in terms of reducing damage to major organs and enhancing antitumor efficacy, and the peptide TD significantly enhanced the delivery of Vem-TD-Lip across the skin. This work provides a new strategy for delivering vemurafenib to target and inhibit subcutaneous melanoma.

A Theoretical Study on Inhibition of Melanoma with Controlled and Targeted Delivery of siRNA via Skin Using SPACE-EGF

Melanoma is a potentially lethal skin cancer with high mortality rate. Recently, the peptide-mediated transdermal delivery of small interference RNA (siRNA) emerges as a promising strategy to treat melanoma by inducing the apoptosis of tumor cells, but the related theoretical model describing the delivery of siRNA under the effect of SPACE-EGF, the growth inhibition of melanoma and the dynamic expanding of the bump on the skin due to the growth of melanoma has not been reported yet. In this article, a theoretical model is developed to describe the percutaneous siRNA delivery mediated by SPACE-EGF to melanoma and the growth inhibition of melanoma. The results present the spatial–temporal distribution of siRNA and the growth of melanoma under the inhibition of siRNA, which shows a good consistency with the experimental results. In addition, this model represents the uplift process of tumors on the skin surface. The model presented here is a useful tool to understand the whole process of the SPACE-EGF-mediated delivery of the siRNA to melanoma through skin, to predict the therapeutic effect, and to optimize the therapeutic strategy, providing valuable references for the treatment of melanoma.