Galactosylated nanostructured lipid carriers for delivery of 5-FU to hepatocellular carcinoma

The aim of the present study was to design a targeted delivery system of 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). Lactobionic acid (LB) was conjugated to stearyl amine (SA) by a chemical reaction. The nanostructured lipid carriers (NLCs), containing LB conjugate, lecithin, glyceryl monostearate, oil [oleic acid (OA) or Labrafac 5 or 10%], and 5-FU, were dissolved in alcohol/acetone, the oil phase was added to the aqueous phase containing Tween 80 or Solutol^® HS15 (0.25 or 0.5%), and NLCs were prepared by an emulsification-solvent diffusion method. Physical properties and drug release were studied in NLCs. The thiazolyl blue tetrazolium bromide assay was used to study the cytotoxicity of NLCs on HepG₂ cells, and the cellular uptake of NLCs was determined by flow cytometry. Fourier transform infrared spectroscopy and ¹H-NMR spectra confirmed the successful conjugation of LB and SA. The optimized NLCs consisted of 0.5% Solutol HS15 and 10% OA oil. The particle size of these nanoparticles was 139.2 nm, with a zeta potential of –18 mV, loading efficiency of 34.2%, release efficiency after 2 hours of the release test was 72.6%, and crystallinity was 0.63%. The galactosylated NLCs of 5-FU were cytotoxic on the HepG₂ cell line in a half concentration of 5-FU and seems promising in reducing 5-FU dose in HCC.     

Galactosylated nanostructured lipid carriers for delivery of 5-FU to hepatocellular carcinoma

The aim of the present study was to design a targeted delivery system of 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). Lactobionic acid (LB) was conjugated to stearyl amine (SA) by a chemical reaction. The nanostructured lipid carriers (NLCs), containing LB conjugate, lecithin, glyceryl monostearate, oil [oleic acid (OA) or Labrafac 5 or 10%], and 5-FU, were dissolved in alcohol/acetone, the oil phase was added to the aqueous phase containing Tween 80 or Solutol(?) HS15 (0.25 or 0.5%), and NLCs were prepared by an emulsification-solvent diffusion method. Physical properties and drug release were studied in NLCs. The thiazolyl blue tetrazolium bromide assay was used to study the cytotoxicity of NLCs on HepG(2) cells, and the cellular uptake of NLCs was determined by flow cytometry. Fourier transform infrared spectroscopy and (1)H-NMR spectra confirmed the successful conjugation of LB and SA. The optimized NLCs consisted of 0.5% Solutol HS15 and 10% OA oil. The particle size of these nanoparticles was 139.2 nm, with a zeta potential of -18 mV, loading efficiency of 34.2%, release efficiency after 2 hours of the release test was 72.6%, and crystallinity was 0.63%. The galactosylated NLCs of 5-FU were cytotoxic on the HepG(2) cell line in a half concentration of 5-FU and seems promising in reducing 5-FU dose in HCC.     

Polyelectrolyte microcapsules as the systems for delivery of biologically active substances

Based on the method of the layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, sodium alginate (Alg) and poly-L-lysine (PLL), novel biodegradable microcapsules have been prepared for delivery of biological active substances (BAS). Porous spherical CaCO₃ microparticles were used as templates. The template cores were coated with several layers of oppositely charged polyelectrolytes forming shell on the core surface. The core-shell microparticles were converted into hollow microcapsules by means of core dissolution with EDTA. Mild conditions for microcapsules preparation allow to perform incorporation of various biomolecules maintaining their bioactivity. Biocompatibility and biodegradability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme retained about 86% of the activity compared to a native chymotrypsin. The resultant microcapsules were stable in acidic medium and could be easily decomposed by trypsin treatment in slightly alkaline medium. Chymotrypsin was shown to be active after its release from the microcapsules decomposed by the trypsin treatment. Thus, the microcapsules prepared by the LbL technique can be used for the development of new type of BAS delivery systems in humans and animals.     

Lecithin soybean phospholipid nano-transfersomes as potential carriers for transdermal delivery of the human growth hormone

Pharmaceutical molecules such as peptides and proteins are usually injected into the body. Numerous efforts have been made to find new noninvasive ways to administer these peptides. In this study, highly flexible vesicles (transfersomes [TFs]) were designed as a new modern transdermal drug delivery system for systemic drug administration through the skin, which had also been evaluated in vitro. In this study, two growth hormone-loaded TF formulations were prepared, using soybean lecithin and two different surfactants; F₁_sodium deoxycholate and F ₂_sodium lauryl sulfate. Thereafter, the amount of skin penetration by the two formulas was assessed using the Franz diffusion cell system. TF formulations were evaluated for size, zeta potential and in vitro skin penetration across the rat skin. Results indicated that vesicle formulations were stable for 4 weeks and their mean sizes were 241.33 ± 17 and 171 ± 12.12 nm in the F ₁ and F ₂ formulation, respectively. After application to rat skin, transport of the human growth hormone (hGH) released from the TF formulations was found to be higher than that of the hGH alone. Maximum amounts of transdermal hormone delivery were estimated to be 489.54 ± 8.301 and 248.46 ± 4.019 ng·cm−2 , for F ₁ and F ₂, respectively. The results demonstrate the capability of the TF-containing growth hormone in transdermal delivery and superiority of the F ₁ to F ₂ TFs.     

Polyelectrolyte complexes of gum kondagogu and chitosan,as diclofenac carriers

Polyelectrolyte complexes (PEC) of gum kondagogu (GKG) and chitosan were prepared by mixing polymeric solutions of different concentrations (0.02–0.18% w/v). The complex formed were loaded with diclofenac sodium, and the release of the drug was measured in vitro and in vivo, along with the measurement of particle size, zeta potential, complex formation, flow properties, and loading efficiency. Maximum yield of PEC was observed at gum kondagogu concentrations above 80%. The PEC showed lower release of diclofenac sodium in 0.1 N HCl as compared to phosphate buffer (pH 6.8). Increasing the concentration of gum kondagogu in PEC led to an increase in drug release. However, PEC 1:3 (gum kondagogu: chitosan) with higher concentration of chitosan showed 98% release with in 4.5 h, owing to the fact that chitosan has a higher degree of swelling in acidic medium. PEC 5:1 and 3:1 showed a 5.3- and 5.8-fold increase in relative bioavailability compared to the free drug when administered orally to the rats.     

Role of growth factor receptor bound protein 7 in hepatocellular carcinoma

The human growth factor receptor-bound protein 7 (Grb7) is an adaptor molecule and is related to cell invasion. In this present study, we investigated the clinical and biological significance of Grb7 expression in human hepatocellular carcinoma (HCC). We reviewed 64 consecutive patients who had undergone liver resection for HCC, and we investigated the correlation between Grb7 expression and clinical outcome. To analyze the biological behavior of Grb7 in vitro and in vivo, we established Grb7 stable knockdown HCC cells using RNA interference technology. The positive staining of Grb7 protein was correlated with portal venous invasion (P < 0.01), hepatic venous invasion (P < 0.01), and intrahepatic metastasis (P < 0.05). Positive expression of Grb7 was significantly correlated with focal adhesion kinase (FAK) protein levels in HCC (P < 0.01). The Grb7- and FAK-positive group showed a significantly poorer prognosis as compared with the Grb7- and FAK-negative group (P < 0.05). Grb7 knockdown HCC cells exhibited significantly lower levels of invasion potential (P < 0.05) and motility (P < 0.05) than the control cells in vitro; moreover, Grb7 knockdown HCC cells showed delayed onset of the tumors compared with the control cells in vivo. Grb7 expression can modulate the invasive phenotype of HCC. Grb7 plays an important role in HCC progression and is strongly associated with expression of FAK. Grb7 could be a therapeutic target in HCC.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.     

Chitosan oligomers as drug carriers for renal delivery of zidovudine

Chitosan oligomers (COS) are water soluble and can be a potential drug carrier for renal targeting delivery. Zidovudine (AZT), a FDA approved antiretroviral drug, has a very short half life and is eliminated very quickly in human plasma and kidney after administration. In this study, AZT-COS conjugates were prepared and evaluated in terms of renal targeting. The in vitro release of AZT from AZT-COS conjugates was confirmed in mice plasma and renal homogenate. The pharmacokinetics study indicated longer mean retention time of AZT-COS conjugates with the values of about 1.5 h than 0.59 h of AZT. The AZT-COS conjugates were found accumulated in kidney other than heart, liver, spleen, lung and brain after i.v. administration, in line with the evidence of the fluorescence imaging of FITC labeled COS in 12 h. In conclusion, AZT-COS conjugates have the potential to be developed into a renal-targeting drug delivery system.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor

Angiogenesis has long been a desired therapeutic approach to improve clinical outcomes of conditions typified by ischemia. Vascular endothelial growth factor (VEGF) has demonstrated the ability to generate new blood vessels in vivo, but trials using intravenous delivery have not yet produced clinical success. Localized, sustained delivery of VEGF has been proven necessary to generate blood vessels as demonstrated by implantable, controlled release devices. Ultimately, nanoparticles delivered by intravenous injection may be designed to accumulate in target tissues and sustain the local VEGF concentration; however, injectable nanosuspensions that control the release of stabilized VEGF must first be developed. In this study, we utilize the heparin binding domain of VEGF to bind the polyanion dextran sulfate, resulting in an enhanced thermal stability of VEGF. Coacervation of the VEGF-bound dextran sulfate with selected polycations (chitosan, polyethylenimine, or poly-L-lysine) produced nanoparticles approximately 250 nm in diameter with high VEGF encapsulation efficiency (50-85%). Release of VEGF from these formulations persisted for >10 days and maintained high VEGF activity as determined by ELISA and a mitogenic bioassay. Chitosan-dextran sulfate complexes were preferred because of their biodegradability, desirable particle size ( approximately 250 nm), entrapment efficiency ( approximately 85%), controlled release (near linear for 10 days), and mitogenic activity.     

Polymeric system for dual growth factor delivery.

The development of tissues and organs is typically driven by the action of a number of growth factors. However, efforts to regenerate tissues (e.g., bone, blood vessels) typically rely on the delivery of single factors, and this may partially explain the limited clinical utility of many current approaches. One constraint on delivering appropriate combinations of factors is a lack of delivery vehicles that allow for a localized and controlled delivery of more than a single factor. We report a new polymeric system that allows for the tissue-specific delivery of two or more growth factors, with controlled dose and rate of delivery. The utility of this system was investigated in the context of therapeutic angiogenesis. We now demonstrate that dual delivery of vascular endothelial growth factor (VEGF)-165 and platelet-derived growth factor (PDGF)-BB, each with distinct kinetics, from a single, structural polymer scaffold results in the rapid formation of a mature vascular network. This is the first report of a vehicle capable of delivery of multiple angiogenic factors with distinct kinetics, and these results clearly indicate the importance of multiple growth factor action in tissue regeneration and engineering.     

Synthesis and biodegradation of nanogels as delivery carriers for carbohydrate drugs

Biodegradable nanogels loaded with rhodamine B isothiocyanate-dextran (RITC-Dx) as a model for water-soluble biomacromolecular drugs were prepared using atom-transfer radical polymerization (ATRP) in a cyclohexane inverse miniemulsion in the presence of a disulfide-functionalized dimethacrylate cross linker. UV-vis spectroscopy was used to characterize the extent of incorporation of RITC-Dx into the nanogels. The loading efficiency of RITC-Dx into the nanogels exceeded 80%. These nanogels were degraded into polymeric sols in a reducing environment to release the encapsulated carbohydrate drugs. The released carbohydrate biomolecules specifically interacted with concanavalin A in water, suggesting that the biodegradable nanogels could be used as carriers to deliver carbohydrate drugs that can be released upon degradation to bind to pathogens based on lectins.     

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.     

Nanostructured lipid carriers as semisolid topical delivery formulations for diflucortolone valerate

Context: Topical treatment of skin disease needs to be strategic to ensure high drug concentration in the skin with minimum systemic absorption.

Objective: The aim of this study was to produce semisolid nanostructured lipid carrier (NLC) formulations, for topical delivery of the corticosteroid drug, diflucortolone valerate (DFV), with minimum systemic absorption.

Method: NLC formulations were developed using a high shear homogenization combined with sonication, using Precirol® ATO5 or Tristearin® as the solid lipid, Capryol? or isopropyl myristate as the liquid lipid and Poloxamer® 407 as surfactant. The present study addresses the influence of different formulations composition as solid lipid, liquid lipid types and concentrations on the physicochemical properties and drug release profile from NLCs.

Results and discussion: DFV-loaded NLC formulations possessed average particle size ranging from 160.40?nm to 743.7?nm with narrow polydispersity index. The encapsulation efficiency was improved by adding the lipid-based surfactants (Labrasol® and Labrafil® M1944CS) to reach 68%. The drug release from the investigated NLC formulations showed a prolonged release up to 12?h. The dermatopharmacokinetic study revealed an improvement in drug deposition in the skin with the optimized DFV-loaded NLC formulation, in contrast to a commercial formulation.

Conclusion: NLC provides a promising nanocarrier system that work as reservoir for targeting topical delivery of DFV.     

Transforming growth factor beta1 and soluble Fas serum levels in hepatocellular carcinoma

In this study we assessed the usefulness of serum Transforming Growth Factor-beta1 (TGF-beta1) and soluble Fas (sFas) in distinguishing liver cirrhosis (LC) with and without hepatocellular carcinoma (HCC) as compared with alpha-fetoprotein (AFP). Serum TGF-beta1 and sFas levels were measured by ELISA in 51 LC patients, 54 patients with HCC and 30 healthy donors. Considering as a cut-off limit (mean+1SD of controls) 74 pg/ml and 637 pg/ml for TGF-beta1 and sFas, respectively, we computed serum concentrations of TGF-beta1 and sFas as a score (mean+/-SD). The positive frequency of serum TGF-beta1 levels in HCC patients (54%) was greater than in LC patients (26%) and healthy donors (3%). TGF-beta1 levels were higher in HCC (1.6+/-0.5) than in LC (1.1+/-0.2) (P<0.0001) and healthy donors (0.6+/-0.2). Using a cut-off limit of 82 pg/ml (mean+2SD), the positive frequency of TGF-beta1 was 20% in HCC patients. None of the controls and LC patients had TGF-beta1 levels higher than 82 pg/ml. The positive frequency of serum sFas levels was 100% in HCC patients, 98% in LC patients and 3% in healthy controls. Serum sFas levels were higher in HCC (2.5+/-0.7) than in LC (1.9+/-0.5) (P<0. 001) and healthy donors (0.6+/-0.3). No significant change of positive frequency was obtained by setting sFas cut-off at higher levels. sFas values did not correlate with TGF-beta1 levels. No relationship was found between TGF-beta1 amounts and AFP levels. However, in the 23% of HCC patients, with normal AFP values TGF-beta1 levels were higher than the cut off. These findings suggest the potential usefulness for TGF-beta1 assay in AFP-negative HCC.     

Epidermal growth factor gene polymorphism and risk of hepatocellular carcinoma: a meta-analysis

Background

Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in the epidermal growth factor (EGF) gene. Previous work suggests an association between the EGF 61*A/G polymorphism (rs4444903) and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent. Therefore, we performed a meta-analysis of several studies covering a large population to address this controversy.

Methods

PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Data were abstracted independently by two reviewers. A meta-analysis was performed to examine the association between EGF 61*A/G polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.

Results

Eight studies were chosen in this meta-analysis, involving 1,304 HCC cases (1135 Chinese, 44 Caucasian and 125 mixed) and 2,613 controls (1638 Chinese, 77 Caucasian and 898 mixed). The EGF 61*G allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.29, 95% CI = 1.16–1.44, p<0.001), homozygote comparison (OR = 1.79, 95% CI = 1.39–2.29, p<0.001) and a recessive genetic model (OR = 1.34, 95% CI = 1.16–1.54, p<0.001), while patients carrying the EGF 61*A/A genotype had significantly lower risk of HCC than those with the G/A or G/G genotype (A/A vs. G/A+G/G, OR = 0.66, 95% CI = 0.53–0.83, p<0.001).

Conclusion

The 61*G polymorphism in EGF is a risk factor for hepatocarcinogenesis while the EGF 61*A allele is a protective factor. Further large and well-designed studies are needed to confirm this conclusion.     

Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.