Yan Zhuang

PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution

Lymph nodes (LNs) are peripheral lymphoid organs essential for vaccine-induced immune responses. Although cationic liposomes have been documented as a novel adjuvant and vaccine delivery system, whether enhancing LN targeting would improve the efficiency of cationic liposome-formulated vaccines has not been elucidated yet. In the present study we investigated the effect of PEGylation on LN targeting and the immunogenicity of cationic liposome-formulated vaccines. DOTAP cationic liposomes were incorporated with 1 or 5mol% of DSPE-PEG2000 and labeled with near infrared fluorescent dyes. The lymphatic trafficking and biodistribution of different liposomes after subcutaneous (s.c.) injection were recorded using an in-vivo imaging system. The results showed that incorporation of 1mol% DSPE-PEG2000 not only accelerated the drainage of DOTAP liposomes into draining LNs, but also prolonged their LN retention and enhanced liposome uptake by resident antigen-presenting cells. On the other hand, although incorporating 5mol% of DSPE-PEG2000 into DOTAP liposomes enhanced their LN retention and uptake to a lesser extent, it prolonged blood circulation of DOTAP liposomes and increased their splenic accumulation. In addition, PEGylated DOTAP liposomes augmented primary and secondary anti-OVA antibody responses more potently than nonPEGylated DOTAP liposomes did. Hence, incorporating a small amount of DSPE-PEG2000 into DOTAP liposomes not only increased the passive LN targeting of DOTAP-formulated vaccines but also modulated their biodistribution in vivo, which consequently improved the efficiency of cationic liposome-formulated vaccines.

Toll-like receptor 3 agonist complexed with cationic liposome augments vaccine-elicited antitumor immunity by enhancing TLR3-IRF3 signaling and type I interferons in dendritic cells.

Cancer vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment. However, its antitumor effect remains unsatisfied due to the poor immunogenicity of tumor antigens (Ags). Although polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, has been reported as a promising adjuvant for cancer vaccines, its immunopotency may be limited by insufficient cellular penetration. In the present study, we incorporated PIC into DOTAP cationic liposome to generate PIC-DOTAP Liposome Complex (PDLC) nanoparticles. The results showed that PDLC was more potent than DOTAP or PIC to enhance vaccine-induced tumor-specific cytotoxic T lymphocyte (CTL) response and IFN-γ production. Moreover, two doses of PDLC vaccines remarkably suppressed tumor growth in mice, which involved the participance of CD8(+) T cells and depended on the presence of Ag. The superior antitumor effect of PDLC vaccines could be attributable to enhanced maturation of mouse bone-marrow dendritic cells (BMDCs) and increased production of type I IFNs. More importantly, PDLC strengthened the TLR3 signaling in BMDCs by enhancing the interaction of PIC with TLR3 and augmenting downstream IRF-3 phosphorylation, as well as elevating IRF-3/IRF-7 mRNA transcription. Taking together, the complex of PIC and DOTAP liposomes enhanced PIC uptake and consequential TLR3 signaling in BMDCs, which in turn promoted DC maturation and type I IFN production, thereby augmenting the antitumor effect of cancer vaccines.

A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

Electrospun Collagen Fibers with Spatial Patterning of SDF1α for the Guidance of Neural Stem Cells

Producing gradients of biological cues into nerve conduits is crucial for nerve guidance and regeneration. Herein, the fabrication of gradients of stromal cell-derived factor-1α (SDF1α) on electrospun collagen mats is reported using an electrohydrodynamic jet printing technique. The fabrication of various SDF1α gradated patterns on collagen fibrous mats is successfully demonstrated including shallow continuous gradient, steep continuous gradient, and step gradient by controlling the processing parameters. The SDF1α graded collagen scaffolds show a long-term stable gradient, as SDF1α is fused with a unique peptide of collagen binding domain (CBD), and CBD-SDF1α can specifically bind to the collagen mat. Such graded scaffolds exhibit sustained release of SDF1α. Further examination of neural stem cell (NSC) response to the CBD-SDF1α gradients with various patterns show that the NSCs can sense the CBD-SDF1α gradients, display a polarized morphology, and tend to migrate toward the region with a higher CBD-SDF1α content. The collagen mats with CBD-SDF1α gradients guide gradual distribution of NSCs, and NSC-differentiated neurons and astrocytes after seeding for 1 and 7 d. This new class of CBD-SDF1α gradient scaffolds can potentially be employed for guided nerve regeneration.

Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds.

To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors.

Radially Aligned Electrospun Fibers with Continuous Gradient of SDF1α for the Guidance of Neural Stem Cells.

Repair of spinal cord injury will require enhanced recruitment of endogenous neural stem cells (NSCs) from the central canal region to the lesion site to reestablish neural connectivity. The strategy toward this goal is to provide directional cues, e.g., alignment topography and biological gradients from the rostral and caudal ends toward the center. This study demonstrates a facile method for fabrication of continuous gradients of stromal-cell-derived factor-1α (SDF1α) embedded in the radially aligned electrospun collagen/poly (ε-caprolactone) mats. Gradients can be readily produced in a controllable and reproducible fashion by adjusting the collection time and collector size during electrospinning. To get a long-term gradient, the SDF1α is fused with a unique peptide of collagen-binding domain (CBD), which can bind to collagen specifically. Aligned CBD-SDF1α gradients show stable, sustained, and gradual release during 7 d. Further, the effect of aligned CBD-SDF1α gradients on the guidance of NSCs is investigated. It is found that the CBD-SDF1α gradient scaffolds direct and enhance NSC migration from the periphery to the center along the aligned electrospun fibers. Taken together, the tubular conduits based on radially aligned electrospun fibers with continuous SDF1α gradient show great potential for guiding nerve regeneration.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins.

Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via Recruiting Endogenous Stem Cells

The reconstruction of bone usually depends on substitute transplantation, which has drawbacks including the limited bone substitutes available, comorbidity, immune rejection, and limited endogenous bone regeneration. Here, we constructed a functionalized bone substitute by combining application of the demineralized bone matrix (DBM) and collagen-binding stromal-cell-derived factor-1α (CBD-SDF-1α). DBM was a poriferous and biodegradable bone substitute, derived from bovine bone and consisting mainly of collagen. CBD-SDF-1α could bind to collagen and be controllably released from the DBM to mobilize stem cells. In a rat femur defect model, CBD-SDF-1α-modified DBM scaffolds could efficiently mobilize CD34+ and c-kit+ endogenous stem cells homing to the injured site at 3 days after implantation. According to the data from micro-CT, CBD-SDF-1α-modified DBM scaffolds could help the bone defects rejoin with mineralization accumulated and bone volume expanded. Interestingly, osteoprotegerin (OPG) and osteopontin (OPN) were highly expressed in CBD-SDF-1α group at an early time after implantation, while osteocalcin (OCN) was more expanded. H&E and Massons trichrome staining showed that the CBD-SDF-1α-modified DBM scaffold group had more osteoblasts and that the bone defect rejoined earlier. The ultimate strength of the regenerated bone was investigated by three-point bending, showing that the CBD-SDF-1α group had superior strength. In conclusion, CBD-SDF-1α-modified DBM scaffolds could promote bone regeneration by recruiting endogenous stem cells.

Lymphatic-targeted cationic liposomes: A robust vaccine adjuvant for promoting long-term immunological memory

Although retaining antigens at the injection site (the so-called “depot effect”) is an important strategy for vaccine development, increasing evidence showed that lymphatic-targeted vaccine delivery with liposomes could be a promising approach for improving vaccine efficacy. However, it remains unclear whether antigen depot or lymphatic targeting would benefit long-term immunological memory, a major determinant of vaccine efficacy. In the present study, OVA antigen was encapsulated with DOTAP cationic liposomes (LP) or DOTAP-PEG-mannose liposomes (LP-Man) to generate depot or lymphatic-targeted liposome vaccines, respectively. The result of in vivo imaging showed that LP mostly accumulated near the injection site, whereas LP-Man not only effectively accumulated in draining lymph nodes (LNs) and the spleen, but also enhanced the uptake by resident antigen-presenting cells. Although LP vaccines with depot effect induced anti-OVA IgG more potently than LP-Man vaccines did on day 40 after priming, they failed to mount an effective B-cell memory response upon OVA re-challenge after three months. In contrast, lymphatic-targeted LP-Man vaccines elicited sustained antibody production and robust recall responses three months after priming, suggesting lymphatic targeting rather than antigen au to depot promoted the establishment of long-term memory responses. The enhanced long-term immunological memory by LP-Man was attributed to vigorous germinal center responses as well as increased Tfh cells and central memory CD4+ T cells in the secondary lymphoid organs. Hence, lymphatic-targeted vaccine delivery with LP-Man could be an effective strategy to promote long-lasting immunological memory.

A novel low-toxic liposomal adjuvant: Different liposome compositions regulate monocyte activation and viability

Liposomes are nanoparticles consisting of phospholipid-bilayer membranes and aqueous compartments. They can effectively incorporate both hydrophobic and hydrophilic molecules, and therefore have been widely applied as a carrier for gene and drug delivery. Moreover, liposomes are considered as a novel vaccine delivery system, which encapsulate both Antigen (Ag) and immunomodulatory agents, facilitating Ag delivery to specific immune cells in vivo. Till date, liposomal vaccines against various diseases, such as cancer, HIV, Hepatitis B and malaria, have been investigated and found to be safe. However, their immunogenicity remains to be further improved [1]. Although conventional liposomes are usually formulated with neutral and/or negatively charged lipids, many studies indicated that the cationic liposomes with positive surface charge more effectively enhanced Ag-specific immune response and promoted vaccine-induced anti-cancer responses than other liposomes [2]. On the other hand, cationic liposomes at a high concentration significantly induced apoptosis of dendritic cells and suppressed immune response, suggesting the immunotoxic effect [3]. In the present study, we incorporated 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a neutral lipid into dioleoyl-3-trimethylammoniumpropane (DOTAP), a cationic liposome, and investigate the effect of different liposome formulations on monocyte activation and viability. Our aim is to develop a safe and effective liposomal adjuvant for vaccine delivery.