Development of Triple-Negative Breast Cancer-Targeted Liposomes with MUC16 Binding Peptide Ligand in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a highly malignant tumor that does not express the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2). As molecular approaches to these targets have limited clinical utility in TNBC, novel strategies for the treatment of TNBC are urgently needed. MUC16 (Mucin-16) is a glycoprotein involved in cell proliferation and apoptosis and is overexpressed in breast cancer. To develop a clinically available strategy for TNBC treatment, we synthesized a MUC16 targeted peptide (EVQ)-grafted lipid derivative, EVQ-(SG)5-lipid, and prepared EVQ-(SG)5/PEGylated liposomes of 100 nm by size and a slightly negative ζ-potential value. Thus, we aimed at investigating the association between EVQ-(SG)5/PEGylated and TNBC cell lines by interacting with MUC16 using an in vitro model. In addition, we aimed at exploring the intracellular distribution and cellular uptake pathway of EVQ-(SG)5/PEGylated liposomes as novel drug delivery carriers for TNBC.

Investigation of enhanced intracellular delivery of nanomaterials modified with novel cell-penetrating zwitterionic peptide-lipid derivatives.

Functionalized drug delivery systems have been investigated to improve the targetability and intracellular translocation of therapeutic drugs. We developed high functionality and quality lipids that met unique requirements, focusing on the quality of functional lipids for the preparation of targeted nanoparticles using microfluidic devices. While searching for a lipid with high solubility and dispersibility in solvents, which is one of the requirements, we noted that KK-(EK)4-lipid imparts nonspecific cellular association to polyethylene glycol (PEG)-modified (PEGylated) liposomes, such as cell-penetrating peptides (CPPs). We investigated whether KK-(EK)4-lipid, which has a near-neutral charge, is a novel CPP-modified lipid that enhances the intracellular translocation of nanoparticles. However, the cellular association mechanism of KK-(EK)4-lipid is unknown. Therefore, we synthesized (EK)n-lipid derivatives based on the sequence of KK-(EK)4-lipid and determined the sequence sites involved in cellular association. In addition, KK-(EK)4-lipid was applied to extracellular vesicles (EVs) and mRNA encapsulated lipid nanoparticles (mRNA-LNPs). KK-(EK)4-lipid-modified EVs and mRNA-LNPs showed higher cellular association and in vitro protein expression, respectively, compared to unmodified ones. We elucidated KK-(EK)4-lipid to have potential for applicability in the intracellular delivery of liposomes, EVs, and mRNA-LNPs.

Synthesis and evaluation of a novel adapter lipid derivative for preparation of cyclic peptide-modified PEGylated liposomes: Application of cyclic RGD peptide.

Peptide ligand modified nanoparticles can simply prepared by post-insertion method to mix pre-formed nanoparticles with peptide-lipid conjugates in an aqueous solution at an optimal temperature. Therefore, water dispersibility of peptide-lipid conjugates is a very important factor for implementing the post-insertion method. We proposed that highly water dispersible peptide-lipid conjugates can be easily synthesized by separately designing novel adapter lipids with different water dispersibility and reacting them with ligands in a highly efficient manner. Adapter lipids have three critical roles; as spacers of ligand-conjugated lipids for efficient ligand presentation, as structures that form discrete molecular weight distributions, and as providing water dispersibility. In this study, we developed a novel adapter-lipid derivative that enables a variety of cyclic peptide modifications using the click reaction. The integrin αvß3-targeted cyclic RGDfK (cRGD) peptide was selected as the cyclic peptide ligand. We designed a novel alkyne-tagged lipid with a discrete peptide spacer and bound the cRGD peptide using a click reaction to synthesize a cRGD-conjugated lipid with good water dispersibility for the preparation of cRGD-modified PEGylated liposomes using the post-insertion method. We also revealed that cRGD-modified PEGylated liposomes are efficiently associated with integrin αvß3-expressing murine colon carcinoma (Colon-26) cells in a modification amount- and peptide sequence-dependent manner, showing high cytotoxicity upon loading with doxorubicin. This novel adapter lipid derivative can be used to synthesize various cyclic peptides by click reactions and will provide useful insights for the future development of cyclic peptide-modified PEGylated liposomes.

Microfluidic Post-Insertion Method for the Efficient Preparation of PEGylated Liposomes Using High Functionality and Quality Lipids.

Introduction: Targeted liposomes using ligand peptides have been applied to deliver therapeutic agents to the target sites. The post-insertion method is commonly used because targeted liposomes can be prepared by simple mixing of ligand peptide-lipid and liposomes. A large-scale preparation method is required for the clinical application of ligand-peptide-modified liposomes. Large-scale preparation involves an increase in volume and a change in the preparation conditions. Therefore, the physicochemical properties of liposomes may change owing to large alterations in the preparation conditions. To address this issue, we focused on a microfluidic device and developed a novel ligand peptide modification method, the microfluidic post-insertion method. Methods: We used integrin αvß3-targeted GRGDS (RGD) and cyclic RGDfK (cRGD)-modified high functionality and quality (HFQ) lipids, which we had previously developed. First, the preparation conditions of the total flow rate in the microfluidic device for modifying HFQ lipids to polyethylene glycol (PEG)-modified (PEGylated) liposomes were optimized by evaluating the physicochemical properties of the liposomes. The targeting ability of integrin αvß3-expressing colon 26 murine colorectal carcinoma cells was evaluated by comparing the cellular association properties of the liposomes prepared by the conventional post-insertion method. Results: When the RGD-HFQ lipid was modified into PEGylated liposomes by varying the total flow rate (1, 6, and 12 mL/min) of the microfluidic device, as the total flow rate increased, the polydispersity index also increased, whereas the particle size did not change. Furthermore, the RGD- and cRGD-modified PEGylated liposomes prepared at a total flow rate of 1 mL/min showed high cellular association properties equivalent to those prepared by the conventional post-insertion method. Conclusion: Microfluidic post-insertion method of HFQ lipids might be useful for clinical application and large-scale preparation of targeted liposomes.

[Development of Nano-DDS Carriers for Control of Spatial Distribution Using Multi-color Deep Imaging].

Because active-targeted liposomes are very complex formulations, quality characteristics of functional lipids have not been defined yet, and this is a major obstacle in clinical application of active targeted liposomes. We have developed high functionality and quality (HFQ) lipids, which define quality characteristics of functional lipids for clinical drug delivery system (DDS) applications. Because HFQ lipids are designed to enable facile and rapid functionalization of DDS carrier by simple and one-step mixing, we are expanding applications for not only liposomes but also exosomes and cells. Recently, we developed multi-color deep imaging by tissue clearing for analysis of spatial distribution of DDS in various tissues. Nanocarriers are usually non-uniformly distributed in solid tumors because of their heterogeneity. Especially, in refractory cancer such as pancreatic cancer, the presence of collagen and blood vessels greatly affects intra-tumor distribution of DDS carrier. Therefore information on spatial relations between the tissue structure and DDS carrier is important to regulate precisely intra-tumor distribution of DDS carrier. Recently, our group has established multi-color deep imaging to analyze spatial distribution of stromal collagen, liposomes, and blood vessels in pancreatic tumor tissue. In this review, we present recent research in developing HFQ lipids. Moreover, current status of research on DDS for pancreatic cancer treatment is reviewed.

Targeted co-delivery of protein and drug to a tumor in vivo by sophisticated RGD-modified lipid-calcium carbonate nanoparticles.

Synchronized bio-distribution of combination therapies has several merits such as synergistic effects and reduced side-effects. Co-delivery of a protein and small molecule drug using a single nanocarrier is challenging because they possess totally different characteristics. Herein, we report the development of sophisticated nanoparticles composed of lipids, calcium carbonate and RGD peptide ligands for the co-delivery of a protein and small molecule drug combination via a simple preparation method. A 'one-step' ethanol injection method was employed to prepare the highly organized nanoparticles. The nanoparticles exhibited a spherical shape with ca. 130 nm diameter, and clearly had an integrated lipid layer covering the periphery. As a ligand, an RGD-modified lipid was post-inserted into the nanoparticles, which was important to overcome the 'PEG dilemma'. The pH-sensitivity of the targeted nanoparticles contributed to the efficient intracellular co-delivery of a protein and drug combination in Colon26 tumor cells, and noticeably improved their accumulation in the tumor region of xenograft mice. Synchronized bio-distribution of the protein and drug was achieved, which was the foundation for the synergistic effects of the combination. The targeting capability of the nanoparticles along with their pH-sensitive drug release and the synchronized bio-distribution of their cargos led to the significant antitumor activity of the SOD and paclitaxel combination in mice. This study provides novel information for the design and preparation of functionalized nanoparticles for the delivery of a protein/drug combination in vivo.

Investigation of Intracellular Delivery of NuBCP-9 by Conjugation with Oligoarginines Peptides in MDA-MB-231 Cells.

Oligoarginines (Rn) are becoming promising tools for the intracellular delivery of biologically active molecules. NuBCP-9, a peptide that induces apoptosis in B-cell lymphoma 2 (Bcl-2)-expressing cancer cells, has been reported to promote the uptake and non-specific cytotoxicity of R8, also called octaarginine. However, it is unknown whether a similar synergistic effect can be seen with other Rn. In this study, we conjugated NuBCP-9 with various Rn (n=8, 10, 12, 14) to investigate and compare their cellular uptake characteristics. In addition, their non-specific cytotoxicity and apoptosis-inducing abilities were evaluated. We found that NuBCP-9 conjugated with Rn enhanced cellular uptake mainly through clathrin-mediated endocytosis and macropinocytosis, and that the uptake pathways were not different from those used by unconjugated Rn. However, the cytotoxicity study showed that NuBCP-9-R12 and NuBCP-9-R14 conjugates enhanced non-specific cytotoxicity. We found that NuBCP-9-R10 conjugate had the highest uptake efficiency and induced correspondingly high levels of apoptosis, while resulting in a tolerable degree of non-specific toxicity.

Development of High-Functionality and -Quality Lipids with RGD Peptide Ligands: Application for PEGylated Liposomes and Analysis of Intratumoral Distribution in a Murine Colon Cancer Model.

High-functionality and -quality (HFQ) lipids have a discrete molecular weight and good water dispersibility and can be produced by solid-phase peptide synthesis. Therefore, HFQ lipids are a promising material for the preparation of ligand-grafted PEGylated liposomes. Recently, we have reported serine-glycine repeated peptides ((SG) n) as a spacer of HFQ lipids and to substitute a conventional PEG spacer. We demonstrated the advantage of using (SG) n spacers for peptide ligand presentation on the liposomal surface in vitro; however, the use of (SG) n spacers in ligand-grafted PEGylated liposomes in vivo has not been validated. The aim of this study was to validate the in vivo targeting ability of HFQ lipid-grafted PEGylated liposomes. We synthesized lipids containing GRGDS (RGD-(SG) n-lipid) to target integrin αvß3 and prepared RGD-(SG) n/PEGylated liposomes. Subsequently, their cellular uptake characteristics in murine colon carcinoma (Colon-26) cells were evaluated. Two-color imaging of liposomes and tumor blood vessels following tissue clearing was performed to examine the spatial intratumoral distribution of liposomes. RGD-(SG)5/PEGylated liposomes were selectively associated with the cells in vitro. In vivo analysis of intratumoral distribution following tissue clearing revealed the superior targeting ability of RGD-(SG)5/PEGylated liposomes compared with that of conventional RGD-PEG2000/PEGylated liposomes for both tumor tissues and tumor blood vessels. We successfully synthesized RGD-HFQ lipids to prepare RGD-grafted PEGylated liposomes for the efficient targeting of integrin αvß3-expressing cells. To the best of our knowledge, this is the first report of the intratumoral distribution of ligand-grafted PEGylated liposomes by two-color imaging following tissue clearing.

Synthesis of high functionality and quality mannose-grafted lipids to produce macrophage-targeted liposomes.

The mannose receptor, which is responsible for tumor invasion, proliferation, and metastasis in the tumor microenvironment, is overexpressed in tumor-associated macrophages. Mannose is commonly applied to PEGylated liposomes in macrophage-targeted cancer therapy. To develop a high functionality and quality (HFQ) lipid for macrophage-targeted liposomes, we designed a novel mannosylated lipid with improved mannose receptor binding affinity using serine-glycine repeats (SG)n. We synthesized Man(S)-(SG)5-SSK-K(Pal)2 using only a fluorenylmethyloxycarbonyl (Fmoc) protecting group solid-phase peptide synthesis method, which produced a high-quality lipid at a moderately good yield. We then prepared Man-(SG)5/PEGylated liposomes using a post-insertion technique to insert Man(S)-(SG)5-SSK-K(Pal)2 into the PEGylated liposomes. In vitro cell investigations revealed that the Man-(SG)5/PEGylated liposomes effectively associated with mouse peritoneal macrophages by interacting with the mannose receptors. The results suggest that we produced a novel high-quality, highly functional mannosylated lipid that is suitable for clinical drug delivery applications.

Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: The effect of serine-glycine repeated peptides as a spacer.

Ligand peptide-grafted PEGylated liposomes have been widely studied for targeted drug delivery systems. Because ligand peptides are commonly grafted using PEG as a spacer on the surface of PEGylated liposomes, the interaction between ligand peptides and their corresponding receptors can be interrupted by steric hindrance of the PEG layer. Therefore, we aimed to develop ligand peptide-lipid derivatives to enhance the targeting efficiency of ligand peptide-grafted PEGylated liposomes, and designed a new ligand peptide-lipid derivatives having serine-glycine repeats (SG)n as a spacer based on the peptide length calculated by PyMol (v0.99). We selected KCCYSL (KCC) as the ligand peptide for binding to human epidermal growth factor receptor-2 (HER2). We synthesized new KCC-(SG)n-lipid derivatives (n=3, 5, 7) and evaluated their cellular association in breast cancer cells. KCC-(SG)n/PEGylated liposomes dramatically increased cellular association on HER2-positive breast cancer cells. The results suggest that KCC can be grafted on the surface of KCC-(SG)n/PEGylated liposomes prepared from KCC-(SG)n-lipid derivatives (n=3, 5, 7). In summary, we succeeded in developing KCC-(SG)n-lipid derivatives for the preparation of ligand peptide-grafted PEGylated liposomes.

Anti-MUC1 Aptamer/Negatively Charged Amino Acid Dendrimer Conjugates for Targeted Delivery to Human Lung Adenocarcinoma A549 Cells.

We previously developed a negatively charged amino acid dendrimer to address the safety concerns associated with the constituent unit of these systems, which resulted in the formation of a sixth-generation glutamic acid-modified dendritic poly(L-lysine) system (KG6E). The aim of this study was to develop a nanocarrier for targeted drug delivery into cancer cells. In this study, we have synthesized a conjugate material consisting of anti-mucin 1 (MUC1) aptamer (anti-MUC1 apt) and KG6E (anti-MUC1 apt/KG6E) for targeted drug delivery to human lung adenocarcinoma A549 cells, which express high levels of the MUC1. The anti-MUC1 apt/KG6E was efficiently internalized by the A549 cells and subsequently transported to the endosomal and lysosomal compartments. In contrast, the cellular association of the sequence scrambled aptamer/KG6E conjugate (scrambled apt/KG6E) was much lower than that of the anti-MUC1 apt/KG6E in A549 cells. These results suggest that our newly developed anti-MUC1 apt/KG6E can be internalized in A549 cells via a MUC1 recognition pathway.