Facile and scalable fabrication of exosome-mimicking nanovesicles through PEGylated lipid detergent-aided cell extrusion.

We report a scalable fabrication method to generate exosome-mimicking nanovesicles (ENVs) by using a biocompatible, cell-binding lipid detergent during cell extrusion. A PEGylated mannosylerythritol lipid (MELPEG) detergent was rationally engineered to strongly associate with phospholipid membranes to increase cell membrane deformability and the corresponding friction force during extrusion and to enhance the dispersibility of ENVs. Compared to cell extrusion without detergent, cell extrusion in the presence of MELPEG increased the ENV production yield by approximately 20 times and cellular protein content per MELPEG-functionalized ENV by approximately 2-fold relative to that of unmodified ENVs. We verified that MELPEG strongly binds to ENV membranes and increases membrane deformability via expansion/swelling while preserving the integrity of the phospholipid bilayer structure. The results highlight that the MELPEG-aided cell extrusion process broadly applies to various cell lines; hence, it could be helpful in the production of ENVs for tissue regeneration, drug delivery, and cancer nanomedicine.

Skin protein-derived peptide-conjugated vesicular nanocargos for selected skin cell targeting and consequent activation.

Several studies have reported that a drug nanocarrier conjugated with ligands having cell binding ability improves drug delivery performance, but multiple cell-targeting and the resultant activation in designated cells has not been investigated yet. This study reports a skin cell multi-targeting vesicular nanocargo system. We selectively conjugated several skin protein-derived cell-targeting peptides (CTPs), including KTTKS, NAP-amide, and Lam332, to amphiphilic polymer-reinforced lipid nanovesicles (PLNVs) to specifically target fibroblasts, melanocytes, and keratinocytes, respectively, through effective association with the corresponding cell membrane receptors. We then showed that CTP-conjugated PLNVs specifically bind to the designated skin cells, even in a mixture of different types of skin cells, eventually leading to skin cell multi-targeting and consequent activation. These results highlight that this CTP-conjugated PLNV system has significant potential for developing an intelligent cellular drug delivery technology for dermatological applications.

Fabrication of cell penetrating peptide-conjugated bacterial cellulose nanofibrils with remarkable skin adhesion and water retention performance.

Bacterial cellulose nanofibrils (BCNFs), possessing excellent biocompatibility as well as hygroscopicity, are receiving high interest as a biomaterial for biomedical and healthcare treatments, since they exert various interactions with tissues after surface modification with biochemicals such as peptides, proteins, and catechols. Herein, we report a BCNF-based skin adhesion system, wherein cell penetrating peptide (CPP)-conjugated BCNFs were employed to enhance the attraction to the skin under wet conditions. For this, we conjugated Bac7, a type of CPP, with the carboxylate of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized BCNFs. We showed that Bac7-conjugated BCNFs exhibited both hydrophobic and electrostatic interactions with the cell membrane, which eventually led to the remarkable adhesion against the skin surface. Furthermore, we demonstrated that such tailored skin attraction played a key role in improving skin water retention.

Cell-penetrating peptide-conjugated lipid/polymer hybrid nanovesicles for endoplasmic reticulum-targeting intracellular delivery.

The endoplasmic reticulum (ER) apparatus is a part of the secretory pathway that transports proteins to the plasma membrane through vesicle trafficking, enabling post-translational modification of the newly synthesized proteins. Several diseases such as inflammation, neurodegenerative disorder, and bipolar disorder are closely associated with dysfunction of the ER stress response. Herein, we present an ER-targeting, intracellular delivery approach that utilized cell-penetrating peptide (CPP)-conjugated lipid/polymer hybrid nanovehicles (LPNVs). For this, we patched Penetratin, a type of CPP, onto the LPNVs with vesicular membranes formulated with poly(ethylene oxide)-b-poly(ε-caprolactone)-b-poly(ethylene oxide) (PEO-b-PCL-b-PEO) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). We found that the Penetratin-conjugated LPNV (LPNVPnt) was readily taken up by cells and showed specific ER-targeting ability, which was comparable to that of LPNVs conjugated with other types of CPPs. Moreover, we observed that remarkable lysosomal escape of the LPNVs occurred due to effective pH buffering with the aid of PEO-b-PCL-b-PEO. These results highlighted that our LPNVPnt system could pave the way for the development of an elaborate drug delivery technology for ER-targeting at the intracellular level.

Enhancing membrane modulus of giant unilamellar lipid vesicles by lateral co-assembly of amphiphilic triblock copolymers.

We report a facile, but robust approach to fabricate structurally stable giant unilamellar vesicles (GUVs), on which a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membrane was made rigid by introducing amphiphilic block polymers. Particularly, we found that lateral co-assembly of an amphiphilic triblock copolymer (ATC) structured with a hydrophobic middle block and long molecular weight (20 K g/mol) hydrophilic end blocks remarkably enhanced the stretching modulus (k) of GUVs. When the membrane composition was optimized, the k value of ATC-hybridized GUVs increased to 6.2 × 108 Pa, which was approximately 10-fold higher than that of DPPC GUVs, thus leading to a much longer half-life. Moreover, we demonstrated that our ATC-hybridized GUVs enabled development of a fascinating vesicular model, which shows great potential as a structurally stable cell membrane mimic.

Fabrication of cell membrane-adhesive soft polymeric nanovehicles for noninvasive visualization of epidermal-dermal junction-targeted drug delivery.

We propose a polymeric nanovehicles (PNVs)-based enhanced transdermal delivery platform. A technical advance can be found in that delivery efficiency is significantly enhanced by effective adhesion of PNVs to the cell membrane, which is characterized noninvasively by using a confocal laser scanning microscopy (CLSM)-based skin visualization technique. To this end, the PNVs with a soft core phase were fabricated through co-assembly of two amphiphilic triblock copolymers, poly(ethylene oxide)-b-poly(ε-caprolactone)-b-poly(ethylene oxide) (PEO-b-PCL-b-PEO) and poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-PPO-b-PEO). The softness of PNVs was tuned successfully, while maintaining the particle size at ∼110 nm, by incorporation of PEO-b-PPO-b-PEO into the PNVs to a volume fraction of 0.3. Through an ex vivo skin penetration test, we showed that transactivating transcriptional activator (TAT)-decorated soft PNVs could not only exert strong adhesion to skin but also increase cellular uptake, leading to a transdermal delivery efficiency that is twice that of a hard PNV control. Moreover, CLSM-based noninvasive visualization of a fluorescent drug probe in the skin showed that the adhesiveness and softness of the PNVs contributed directly to the enhancement of transdermal delivery.