Targeting of nanoparticles to cell adhesion molecules for potential immune therapy

Cell adhesion molecules including leukocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) play an important role in regulating inflammatory responses. For circulating leukocytes to enter inflamed tissue or peripheral lymphoid organs, the cells must adhere to and transmigrate between endothelial cells lining blood vessel wall by binding of LFA-1 on leukocytes to ICAM-1 on endothelial cells. In addition, interaction of LFA-1 expressed on T cells and ICAM-1 expressed on antigen presenting cells (APCs) is crucial for immunological synapse formation and hence T cell activation. Clustering of LFA-1 and ICAM-1 by multivalent ligands increases binding avidity of these cell adhesion molecules. In this thesis, multivalent ICAM-1 or LFA-1 ligands were conjugated to the surface of polymeric nanoparticles (NPs) to target the clustering of these receptors and increase the avidity of binding to ICAM-1 or LFA-1. Polymeric nanoparticles possess some advantages over other multivalent ligands since drugs can be protected and released. This delivery system permits modification of the nanoparticle surface without compromising the activity of the drug carried. In chapter 2 of this thesis, a peptide ligand targeting ICAM-1 (cLABL) was conjugated to poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cellular uptake of cLABL conjugated NPs (cLABL-NPs) by lung carcinoma epithelial cells upregulating ICAM-1 was significantly more rapid than control NPs. The specificity of ICAM-1 mediated internalization was confirmed by blocking the uptake of cLABL-NPs to ICAM-1 using free cLABL peptide. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells and provided sustained release of doxorubicin. In chapter 3, a peptide ligand targeting LFA-1 (cIBR) was conjugated to PLGA NPs to specifically target T cells expressing LFA-1. The specificity of NPs targeting LFA-1 was demonstrated by competitive inhibition using free cIBR peptide or by using the I domain of LFA-1 to inhibit the binding of cIBR-NPs. In addition, T-cell adhesion to epithelial cells was inhibited by cIBR-NPs. In chapter 4, nanoparticles capable of blocking LFA-1/ICAM-1 interaction were then studied as inhibitors of T cell conjugation to DCs. LABL-NPs and cIBR-NPs rapidly bound to DCs and inhibited T cell conjugation to DCs to a greater extent than the free peptides, unconjugated NPs, anti-ICAM-1 antibodies and anti-LFA-1 antibodies. In addition, DCs treated with NPs or with cIBR-NPs stimulated the proliferation of T cells, but DCs treated with LABL-NPs did not stimulate T cell proliferation. LABL-NPs and cIBR-NPs also altered cytokine production compared to free ligands suggesting these NPs may offer a unique tool for shaping T cell response. In chapter 5, multivalent ligands having both ovalbumin (OVA) antigen and LABL peptide grafted to hyaluronic acid (HA) were found to bind professional APCs and may offer an alternative targeting approach for inducing immune tolerance. Collectively, results verified that cyclic and linear LABL and cIBR peptides can target NPstoICAM-1 and LFA-1, respectively, to deliver encapsulated agent or to provide function as potent immune modulators

Pharmacognostic Investigation of the Leaves of Mentha cordifolia and Its DNA Fingerprints

Objective: To perform microscopic investigation of Mentha cordifolia leaf and determine DNA fingerprint of the plant.Methods: Tissues of M. cordifolia, M. arvensis var piperascens and M. spicata were investigated under light microscope. DNA marker for M. cordifolia was established by amplification of the internal transcribed spacer (ITS) of nuclear ribosomal RNA gene using conserved plant sequences as primers and fragmentation with the restriction enzyme Bsm1.Results: The results showed the presence of uniseriate epidermal cells covered by a fine cuticle layer, glandular trichomes of multicellular type, capitate and peltate, and non-glandular trichomes. The fragmentation pattern of ITS of nuclear ribosomal RNA gene was applied as DNA marker for discrimination of M. cordifolia from M. arvensis var piperascens and M. spicata.Conclusion: Pharmacognostic investigation of M. cordifolia leaf exhibited the characteristic of Mentha species and the plant is distinguishable from M. arvensis var piperascens and M. spicata by DNA fingerprint pattern.Keywords: Mentha cordifolia, microscopic investigation, DNA marker, internal transcribed space

cIBR effectively targets nanoparticles to LFA-1 on acute lymphoblastic T cells

Leukocyte function associated antigen-1 (LFA-1) is a primary cell adhesion molecule of leukocytes required for mediating cellular transmigration into sites of inflammation via the vascular endothelium. A cyclic peptide, cIBR, possesses high affinity for LFA-1 and conjugation to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles can specifically target and deliver the encapsulated agents to T cells expressing LFA-1. The kinetics of targeted nanoparticle uptake by acute lymphoblastic leukemia T cells was investigated by flow cytometry and microscopy and compared to untargeted nanoparticles. The specificity of targeted nanoparticles binding to the LFA-1 integrin was demonstrated by competitive inhibition using free cIBR peptide or using the I domain of LFA-1 to inhibit the binding of targeted nanoparticles. The uptake of targeted nanoparticles was concentration and energy dependent. The cIBR-conjugated nanoparticles did not appear to localize with lysosomes whereas untargeted nanoparticles were detected in lysosomes in 6 hrs and steadily accumulated in lysosomes for 24 hrs. Finally, T-cell adhesion to epithelial cells was inhibited by cIBR-nanoparticles. Thus, nanoparticles displaying the cIBR ligand may offer a useful targeted drug delivery system as an alternative treatment of inflammatory diseases involving transmigration of leukocytes

Nanoparticles Targeting Dendritic Cell Surface Molecules Effectively Block T cell Conjugation and Shift Response

Dendritic cells (DCs) are potent professional antigen presenting cells (APC) that activate naïve T cells. Interaction of ICAM-1 and LFA-1 molecules on each cell is required for T cell conjugation to DCs which leads to naïve CD4+ T cell activation and proliferation. Nanoparticles capable of blocking LFA-1/ICAM-1 interaction were studied as inhibitors of T cell conjugation to DCs. Primary DCs were primed with ovalbumin, then treated with a peptide that binds ICAM-1 (LABL), a peptide that binds LFA-1 (cIBR) or the same peptides covalently linked to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles (NPs). LABL-NPs and cIBR-NPs rapidly bound to DCs and inhibited T cell conjugation to DCs to a greater extent than the free peptides, unconjugated nanoparticles (NPs), anti-ICAM-1 antibodies and anti-LFA-1 antibodies. In addition, DCs treated with NPs or with cIBR-NPs stimulated the proliferation of T cells, but DCs treated with LABL-NPs did not stimulate T cell proliferation. Nanoparticles targeting ICAM-1 or LFA-1 also altered cytokine production by DC cocultured with T cells when compared to free ligands suggesting these NPs may offer a unique tool for shaping T cell response

Hyaluronic acid graft polymers displaying peptide antigen madulate dendritic cell response in vitro

A novel oxime grafting scheme was utilized to conjugate an ICAM-1 ligand (LABL), a cellular antigen ovalbumin (OVA), or both peptides simultaneously to hyaluronic acid (HA). Samples of HA only and the various peptide grafted HA were found to bind to dendritic cells (DCs). HA with grafted LABL and OVA showed the greatest binding to DCs. Dendritic cells treated with HA, HA with grafted LABL, or HA with grafted LABL and OVA, significantly suppressed T cell and DC conjugate formation, T cell proliferation and reduced proinflammatory cytokine production compared to untreated cells. These results suggest that HA serves as an effective backbone for multivalent ligand presentation for inhibiting T cell response to antigen presentation. In addition, multivalent display of both antigen and an ICAM-1inhibitor (LABL) may enhance binding to DCs and could potentially disrupt cellular signaling leading to autoimmunity

ICAM-1 Targeting of Doxorubicin-Loaded PLGA Nanoparticles to Lung Epithelial Cells

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphoctytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been indentified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic® surfactant on poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1 mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system

LFA-1 on Leukemic Cells as a Target for Therapy or Drug Delivery

Leukemia therapeutics are aiming for improved efficacy by targeting molecular markers differentially expressed on cancerous cells. Lymphocyte function-associated antigen-1 (LFA-1) expression on various types of leukemia has been well studied. Here, the role and expression of LFA-1 on leukemic cells and the possibility of using this integrin as a target for drug delivery is reviewed. To support this rationale, experimental results were also included where cIBR, a cyclic peptide derived from a binding site of LFA-1, was conjugated to the surface of polymeric nanoparticles and used as a targeting ligand. These studies revealed a correlation of LFA-1 expression level on leukemic cell lines and binding and internalization of cIBR-NPs suggesting a differential binding and internalization of cIBR-NPs to leukemic cells overexpressing LFA-1. Nanoparticles conjugated with a cyclic peptide against an accessible molecular marker of disease hold promise as a selective drug delivery system for leukemia treatment

High efficiency in vitro wound healing of dictyophora indusiata extracts via anti-inflammatory and collagen stimulating (MMP-2 inhibition) mechanisms

Dictyophora indusiata or Phallus indusiatus is widely used as not only traditional medicine, functional foods, but also, skin care agents. Biological activities of the fruiting body from D. indusiata were widely reported, while the studies on the application of immature bamboo mushroom extracts were limited especially in the wound healing effect. Wound healing process composed of 4 stages including hemostasis, inflammation, proliferation, and remodelling. This study divided the egg stage of bamboo mushroom into 3 parts: peel and green mixture (PGW), core (CW), and whole mushroom (WW). Then, aqueous extracts were investigated for their nucleotide sequencing, biological compound contents, and wound healing effect. The anti-inflammatory determination via the levels of cytokine releasing from macrophages, and the collagen stimulation activity on fibroblasts by matrix metalloproteinase-2 (MMP-2) inhibitory activity were determined to serve for the wound healing process promotion in the stage 2-4 (wound inflammation, proliferation, and remodelling of the skin). All D. indusiata extracts showed good antioxidant potential, significantly anti-inflammatory activity in the decreasing of the nitric oxide (NO), interleukin-1 (IL-1), interleukin-1 (IL-6), and tumour necrosis factor-α (TNF-α) secretion from macrophage cells (p < 0.05), and the effective collagen stimulation via MMP-2 inhibition. In particular, CW extract containing high content of catechin (68.761 ± 0.010 mg/g extract) which could significantly suppress NO secretion (0.06 ± 0.02 µmol/L) better than the standard anti-inflammatory drug diclofenac (0.12 ± 0.02 µmol/L) and their MMP-2 inhibition (41.33 ± 9.44%) was comparable to L-ascorbic acid (50.65 ± 2.53%). These findings support that CW of D. indusiata could be an essential natural active ingredient for skin wound healing pharmaceutical products

Effects of Quercetin and Curcumin Combination on Antibacterial, Antioxidant, In Vitro Wound Healing and Migration of Human Dermal Fibroblast Cells

Wound healing impairment due to a postponed, incomplete, or uncoordinated healing process has been a challenging clinical problem. Much research has focused on wound care, particularly on discovery of new therapeutic approaches for acute and chronic wounds. This study aims to evaluate the effect of the combination of quercetin and curcuminoids at three different ratios on the antimicrobial, antioxidant, cell migration and wound healing properties. The antioxidant activities of quercetin, curcuminoids and the mixtures were tested by DPPH and ABTS free radical scavenging assays. The disc diffusion method was performed to determine the antibacterial activities of quercetin, curcuminoids and the mixtures against S. aureus and P. aeruginosa. The cytotoxicity and cell migratory enhancing effects of quercetin, curcuminoids and the mixtures against human dermal fibroblasts were investigated by MTT assay, scratch assay and Transwell migration assay, respectively. The results showed the synergism of the quercetin and curcuminoid combination to inhibit the growth of S. aureus and P. aeruginosa, with the inhibition zone ranging from 7.06 &plusmn; 0.25 to 8.78 &plusmn; 0.38 mm, respectively. The DPPH free radical scavenging assay demonstrated that the combination of quercetin and curcuminoids yielded lower IC50 values (15.38&ndash;23.70 &micro;g/mL) than curcuminoids alone (25.75 &micro;g/mL). Quercetin and a 3:1 quercetin/curcuminoid mixture at non-toxic concentrations showed the ability to stimulate the migration of fibroblasts across the matrix, whereas only quercetin alone accelerated the wound closure of fibroblasts. In conclusion, the mixture of quercetin and curcuminoids at a 3:1 ratio was the best formulations for use in wound healing due to the antimicrobial, antioxidant and cell-migration-enhancing activities