Current Drug Delivery (v.13, #4)

Meet Our Editorial Board Member by Bin He (481-481).

Cubosomes: Innovative Nanostructures for Drug Delivery by Aindrilla S. Duttagupta, Harsiddhi M. Chaudhary, Kisan R. Jadhav, Vilasrao J. Kadam (482-493).
Background: Some amphiphilic lipids can self-assemble to form bicontinuous cubic liquid crystalline materials in aqueous media. These cubic structures have gained considerable attention since they impart unique properties of practical interest. Cubosomes, being dispersions of an inverted type bicontinuous cubic phase, separate two continuous aqueous regions with a lipid bilayer having the propensity to incorporate drugs of varying polar characteristics. These novel versatile materials possess the properties to form a section of the next generation of advanced biocompatible nanoparticles.
Methods: This review chiefly considers the scope and importance of cubosomes as a proficient drug delivery vehicle. In addition, it also takes into account the various methods of preparation, the drug loading and release behavior as well as different methods of characterization. Their current advances in various arenas ranging from sustained drug release, burn management, melanoma therapy, vaccine delivery, protein delivery, cosmeceutical and theranostic applications are briefly summarized in this overview.
Results: The drug release from cubosomal dispersions have shown enhancement in bioavailability by solubilisation of poorly water soluble drugs, decrease in adverse effects, enhancement of intracellular penetration, protection against degradation, possibility of sustained drug release and the biodegradable nature of lipids is an added advantage.
Conclusion: Recognizing the desirable properties of cubosomes, it has been proposed as a novel carrier for drug delivery systems. Their unique solubilizing, encapsulating, transporting and protecting capabilities make them an attractive vehicle for numerous in vivo drug delivery routes.

Biodegradable Polymeric Nanoparticles as the Delivery Carrier for Drug by Kai Zhao, Dan Li, Ci Shi, Xueling Ma, Guangu Rong, Hong Kang, Xiaohua Wang, Bin Sun (494-499).
Background: Drug research and development has entered into the new epoch of innovation formulation, and the drug delivery system has been in the forefront of pharmaceutical innovation. Nanotechnology is widely used in fiber and textiles, electronics, space, agriculture, forensic science and medical therapeutics. It increasingly plays a significant role in drug delivery system. Compared with traditional delivery system, the nanoparticle drug delivery system has lots of merits, such as the high drug loading ability, the excellent biocompatibility, low toxicity, controlled and targeted drug release.
Methods: We undertook a structured research of biodegradable polymeric nanoparticles used as delivery carrier for drug using a focused review question and inclusion/exclusion criteria. We have searched the bibliographic databases for peerreviewed research literature. The outstanding characteristics of the screened papers were described respectively, and a systematic content analysis methodology was used to analysis the findings.
Results: Seventy-three papers were included in the review, the majority defined leadership and governance approaches that had impacted upon the polymeric nanoparticles as the delivery carrier for drug in therapeutic applications and developments. Seven papers outlined the superiority characteristics of polymeric nanoparticles that applied in the field of vaccine. Forty-seven papers overviewed the application prospects of polymeric nanoparticles used as drug delivery carrier for cancer. These included current advances in research and clinical applications of polymeric nanoparticles. The review identified the drug delivery carrier of biodegradable polymeric nanoparticles, and we described the synthesis methods, applications and challenges of polymeric nanoparticles.
Conclusion: The findings of this review identified that the biodegradable polymeric nanoparticles were used as delivery carrier for drug currently. It also indicates that the biodegradable polymeric nanoparticles play an important role in the drug delivery.

Hyaluronan/Hyaladherins - a Promising Axis for Targeted Drug Delivery in Cancer by Dragana Nikitovic, Katerina Kouvidi, Rafaela-Maria Kavasi, Aikaterini Berdiaki, George N. Tzanakakis (500-511).
Background: Hyaluronan (HA), a glycosaminoglycan, is a key extracellular matrix (ECM) component, and has been established to contribute to fibrotic, angiogenic, inflammatory as well as processes supporting cancer development. The changes in HA deposition in different tumors have been widely studied. Indeed, a multitude of reports demonstrate that HA expression is increased in different neoplasmatic tissues including lung, colon, prostate and breast cancer. The aims of this paper are to critically and in depth discuss aspects of HA metabolism in cancer and recent developments of its utilization in cancer therapy.
Methods: Up to date research and online content are reviewed.
Results: The cellular roles of HA are perpetrated through molecular interactions with HA-binding proteins, called hyaladherins, including CD44 receptor as well as receptor for hyaluronan-mediated motility (RHAMM). HA binding can be followed by receptor-mediated endocytosis. Importantly, hyaladherins show an altered expression in tumor tissues. Indeed, post-translational alterations in CD44 structure have been suggested to regulate the equilibrium between the “inactive” low affinity state and the “active” high affinity state of the HA binding capacity. In this concept HA fragments can be utilized as specific targeting ligands for efficient and safe drug delivery in cancer.
Conclusion: HA-drug bioconjugates and nanoparticles have emerged as a promising platform for drug delivery during cancer treatment as demonstrated in various pre-clinical studies. Recent developments from clinical trials indicate that the utilization of specific HA-drug bioconjugates might be approved for the medical practice in the nearest future.

Background: The purpose of this study was to investigate Quality by Design (QbD) principle for the preparation of hydrogel products to prove both practicability and utility of executing QbD concept to hydrogel based controlled release systems. Product and process understanding will help in decreasing the variability of critical material and process parameters, which give quality product output and reduce the risk. This study includes the identification of the Quality Target Product Profiles (QTPPs) and Critical Quality Attributes (CQAs) from literature or preliminary studies.
Methods: To identify and control the variability in process and material attributes, two tools of QbD was utilized, Quality Risk Management (QRM) and Experimental Design. Further, it helps to identify the effect of these attributes on CQAs. Potential risk factors were identified from fishbone diagram and screened by risk assessment and optimized by 3-level 2- factor experimental design with center points in triplicate, to analyze the precision of the target process. This optimized formulation was further characterized by gelling time, gelling temperature, rheological parameters, in-vitro biodegradation and in-vitro drug release.
Results: Design space was created using experimental design tool that gives the control space and working within this controlled space reduces all the failure modes below the risk level.
Conclusion: In conclusion, QbD approach with QRM tool provides potent and effectual pyramid to enhance the quality into the hydrogel.

Targeting Mechanism of a Novel Liver-targeting Interferon IFN-CSP Involves Liver Heparan Sulfate Proteoglycan by Xuemei Lu, Jie Wang, Xiaobao Jin, Yanting Huang, Wenting Zeng, Jiayong Zhu (528-533).
Background: In our previous study, a novel liver-targeting interferon (IFN-CSP) combining IFN ?2b with plasmodium region I-plus peptide was successfully designed and prepared with Escherichia coli expression systems. The purified IFN-CSP showed anti-HBV activity and liver-targeting potentiality. The present investigation was designed to investigate the molecular mechanisms responsible for liver-targeting of IFN-CSP.
Methods: The binding site of IFN-CSP in hepatocytes was assayed by immunofluorescent staining. The correspondence of HSPG distribution and the pattern of IFN-CSP binding in liver tissue were determined using a confocal laser scanning microscope. Both the hepatocytes and liver tissue were using as model to investigate the effect of enzyme and soluble glycosaminoglycan on IFN-CSP binding using flow cytometry and fluorescence microscope.
Results: Studies of hepatocytes demonstrated that the localization of IFN-CSP in hepatocytes was the plasma membrane. Studies of liver tissue slices showed that IFN-CSP bound to liver tissue in a pattern similar to the distribution of heparan sulfate proteoglycan (HSPG) immunoreactivity. Pretreatment of hepatocytes and liver slices with heparinase reduced the binding of IFN-CSP to HepG2.2.15 cells and liver slices. Coincubation of IFN-CSP with heparin markedly inhibited IFNCSP binding to HepG2.2.15 cells and liver slices.
Conclusion: These results indicate that the molecular mechanisms responsible for IFN-CSP targeting involve binding to HSPG of hepatocytes and liver.

Background: 6-MP has short elimination time (<2 h) and low bioavailability (~ 50%). Present study was aimed to develop time controlled and site targeted delivery of 6-Mercaptopurine (6-MP) for treatment of colon diseases.
Methods: Compression coating technique was used. 32 full factorial design was designed for optimization of the outer coat for the core tablet. For outer coat amount of Eudragit RS 100 and hydroxypropyl methylcellulose (HPMC K100) were employed as independent variables each at three levels while responses evaluated were swelling index and bursting time. Direct compression method was used for tablets formulation.
Results: 80% w/w of microcrystalline cellulose and 20% w/w of croscarmellose sodium were found to be optimum concentration for the core tablet. The outer coat of optimized batch (ED) contains 21.05% w/w Eudragit RS 100 and 78.95% w/w HPMC K100 of total polymer weight. In-vitro dissolution study indicated that combination of polymer retards the drug release in gastric region and releases ?95% of drug in colonic region after ?7 h. Whereas in case of in-vivo placebo x-ray imaging study had shown that the tablet reaches colonic part after 5±0.5 h providing the proof of arrival in the colon. Stability study indicated that the optimized formulation were physically and chemically stable.
Conclusion: Present research work concluded that compression coating by Eudragit RS 100 and HPMC K100 to 6-MP core provides potential colon targeted system with advantages of reduced gastric exposure and enhanced bioavailability. Formulation can be considered as potential and promising candidate for the treatment of colon diseases..

Pharmacokinetics, Organ Toxicity and Antitumor Activity of Docetaxel Loaded in Folate Targeted Cholesterol Based Micelles by Somayeh Taymouri, Jaleh Varshosaz, Farshid Hassanzadeh, Shaghayegh H. Javanmard, Parvin Mahzouni (545-556).
Background: To overcome insufficient concentration of chemotherapeutic drugs at tumor site and severe side effects in non-targeted tissues which limit their use targeting their overexpressed receptors represent a promising approach for cancer therapy.
Methods: The antitumor activity of docetaxel (DTX) loaded in folate targeted Synpronic F127- Cholesterol (FA-PF127-Chol) nanomicelles was evaluated in C57BL6 mice bearing melanoma and their survival was studied. The pharmacokinetic of DTX loaded FA-PF127-Chol micelles in comparison with Taxoter® was investigated in male Wistar rats. The tumor proliferation was detected by Ki67 assay. The systemic organ toxicity was evaluated in healthy bulb-c mice.
Results: DTX loaded FA-PF127-Chol micelles significantly inhibited tumor growth and enhanced animal survival compared to Taxoter® and non-targeted micelles. FA-PF127-Chol micelles significantly enhanced mean residence time (MRT) and AUC0-? of DTX compared to Taxoter®. The immunehistochemical study demonstrated that DTX loaded FA-PF127-Chol significantly inhibited intra-tumoral cell proliferation in comparison with other treated groups. Safety evaluation showed no toxicity of DTX loaded targeted micelles on blood cells. Histopathology analysis of major organs of mice treated with DTX loaded FA-PF127-Chol micelles showed less tissue damages compared to Taxoter® and non-targeted ones.
Discussion: The results of this contribution showed the potential of DTX loaded FA-PF127-Chol in treatment of melanoma.

Nose to Brain Delivery of Midazolam Loaded PLGA Nanoparticles: In Vitro and In Vivo Investigations by Deepak Sharma, Rakesh Kumar Sharma, Aseem Bhatnagar, Dhruv K. Nishad, Thakuri Singh, Reema Gabrani, Sanjeev K. Sharma, Javed Ali, Shweta Dang (557-564).
Objectives: The present study is aimed to develop poly(D, L-lactide-co-glycolic acid) (PLGA) nanoparticles (NP) loaded with midazolam (Mdz) for nose to brain delivery.
Materials and Methods: NP were formulated by nanoprecipitation and characterized for z-average, zeta potential, % drug entrapment and ex vivo drug release. Mdz NP (MNP) were radiolabeled with technetium-99m. Biodistribution and gamma scintigraphic studies were performed on Sprague-Dawley rats following intranasal (i.n) and intravenous (i.v) administration to trace the transport of Mdz for nose-to-brain delivery.
Results and Discussion: MNP showed z-average of 164±4.5nm with polydispersity index 0.099±0.02 and zeta potential of -16.6±2.5mV. Ex vivo drug studies indicated that MNP showed 29±1.2% of permeation upto 4h via sheep nasal mucosa, whereas Mdz suspension (MS) showed drug release of 83±1.2% within 4h. Comparing i.n administration of MNP, MS and i.v administration of MS, scintigraphy imaging and Brain/blood uptake ratios indicated higher brain targeting via i.n administration of MNP.
Conclusion: Results indicated that the i.n MNP could be employed as a non invasive mode of delivery system with improved drug entrapment, stability and controlled drug release over a period of time.

Optimization of Melatonin Dissolution from Extended Release Matrices Using Artificial Neural Networking by Martarelli D., Casettari L., Shalaby K.S., Soliman M.E., Cespi M., Bonacucina G., Fagioli L., Perinelli D.R., Lam J.K.W., Palmieri G.F. (565-573).
Background: Efficacy of melatonin in treating sleep disorders has been demonstrated in numerous studies. Being with short half-life, melatonin needs to be formulated in extended-release tablets to prevent the fast drop of its plasma concentration. However, an attempt to mimic melatonin natural plasma levels during night time is challenging.
Methods: In this work, Artificial Neural Networks (ANNs) were used to optimize melatonin release from hydrophilic polymer matrices. Twenty-seven different tablet formulations with different amounts of hydroxypropyl methylcellulose, xanthan gum and Carbopol®974P NF were prepared and subjected to drug release studies. Using dissolution test data as inputs for ANN designed by Visual Basic programming language, the ideal number of neurons in the hidden layer was determined trial and error methodology to guarantee the best performance of constructed ANN.
Results: Results showed that the ANN with nine neurons in the hidden layer had the best results. ANN was examined to check its predictability and then used to determine the best formula that can mimic the release of melatonin from a marketed brand using similarity fit factor.
Conclusion: This work shows the possibility of using ANN to optimize the composition of prolonged-release melatonin tablets having dissolution profile desired.

In Vitro Evaluation of pH Responsive Doxazosin Loaded Mesoporous Silica Nanoparticles: A Smart Approach in Drug Delivery by Arijit Guha, Nikhil Biswas, Kaustav Bhattacharjee, Piu Das, Ketousetuo Kuotsu (574-581).
Objective: To develop a pH responsive drug delivery system (DDS) for controlled release of therapeutic cargo, Doxazosin Mesylate (DZM) which was loaded into carrier material mesoporous silica nanoparticle (MSN) and subsequently coated with Eudragit S-100(ES-100) to release the drug at pH 7.4.
Material and Methods: We have synthesized cylindrical MSN under acidic condition using non-ionic surfactant (Pluronic® P 123) and Tetraethoxysilane (TEOS). After post synthesis treatment (PST) surfactant was removed by calcination. To obtain pH sensitive release calcined MSN was coated with ES-100 (MSN-DZMES100). The Brauner-Emmett-Teller (BET) surface area, adsorption isotherm, t-plot, pore volume of MSN were done in surface area analyzer to characterize different MSN samples (as synthesized, calcined, and coated).
Result and Discussion: Highest surafce area (427.114 m2/g) was observed in case of calcined sample when compared to as synthesized (3.1198m2/g) and coated MSN (8.8480m2/g). Adsorption pore width of final coated sample was 12.58 nm whereas as synthesized and calcined samples possessed pore width 36.82 nm and 7.32 nm respectively. All uncoated and coated MSN samples were further characterized with FESEM, TEM, FTIR. No significant interaction between drug and MSN was found from FTIR studies. The drug loading into coated mesoporous support was found to be 43.7%. In vitro studies were done at different pH using Franz-diffusion cell. Results showed significant release at pH 7.4 from MSNDZM- ES100. Cumulative drug release over a period of 10 hr was 81% at this systemic pH.
Conclusion: ES-100 coated mesoporous silica nanoparticle is a smart carrier for pH responsive release of guest molecule.

Improving the Efficiency and Safety of Aspirin by Complexation with the Natural Polysaccharide Arabinogalactan by Mikhail V. Khvostov, Tatjana G. Tolstikova, Sergey A. Borisov, Natalja A. Zhukova, Alexander V. Dushkin, Yulia S. Chistyachenko, Nikolay E. Polyakov (582-589).
Background: The main undesirable side effect of the aspirin is the damage to the gastrointestinal mucosa, leading to the formation of erosions, peptic ulcers, and as a result, bleeding. To overcome this problem “host-guest” complexation with natural polysaccharide arabinogalactan could be applied.
Methods: The complex with a weight ratio of ASA:AG = 1:10 was prepared by solid phase method in a rotary mill. Complex was administered orally to mice or rats at doses of 250, 500 or 1000 mg/kg. The “acetic acid induced writhing” and “hot plate” tests were used as an in vivo pain models. The antiinflammatory activity was studied using “histamine swelling” test. Also, long-term (30 days) oral introduction of the complex to rats was performed and gastric mucosa damages were evaluated. In all experiments pure aspirin (ASA) was used as a control in appropriate doses.
Results: The minimal effective analgesic dose of the complex was 250 mg/kg, equivalent to 23 mg/kg of ASA, a dose in which aspirin itself was not active. The anti-inflammatory effect was found at relatively higher doses: 500 and 1000 mg/kg (46 and 92 mg/kg of ASA respectively) for the complex and only at 100 mg/kg for the ASA. Long-term introduction of the complex at doses of 250 and 500 mg/kg was safe for gastric mucosa, while ASA at the dose of 50 mg/kg showed a strong gastric mucosal damage.
Conclusion: The effective analgesic and anti-inflammatory doses of 1:10 aspirin complex with arabinogalactan are twice less compared to pure aspirin and safer for the gastrointestinal mucosa.

Background: Choroidal neovascularization (CNV), also known as subretinal neovascularization, causes serious damage to the central vision as it happens more commonly in macula. The most important factor involved in angiogenesis is vascular endothelial growth factor (VEGF). By an RNAi technique, VEGF gene knockdown can be used to treat CNV. PEG-conjugated poly (amidoamine) (PEGPAMAM) dendrimers as a new type of synthetic polymers are very promising to be gene delivery carriers.
Methods: To investigate siRNA delivery efficacy of PEG-PAMAM dendrimers, we prepared dendriplexes of PEG-PAMAM dendrimers with a fluorescence-labelled siRNA (PEG-PAMAM/FAM siRNA) or VEGF siRNA (PEG-PAMAM/VEGF siRNA), and studied transfection and downregulation efficacy of the dendriplexes in a cobalt chloride (CoCl2)-induced neovascularization model in retinal vascular endothelial cells (RF/6A).
Results: Our results demonstrate that PEG-PAMAM dendrimers had significantly higher transfection efficiency to FAM siRNA than a commercial transfection reagent PEI (1.4-fold, P<0.001) measured by flow cytometry. Compared to the PEI/VEGF siRNA polyplexes, the dendriplexes of the PEG-PAMAM/VEGF siRNA more significantly downregulated VEGF gene expression (P < 0.01) at both mRNA and protein expression level. A tube formation assay also proved that the PEG-PAMAM/VEGF siRNA dendriplexes more significantly inhibited vascular-like formation than PEI/VEGF siRNA did (P < 0.001) in RF/6A.
Conclusion: This study demonstrated that G5-PEG was more efficient than PEI in facilitating siRNA delivery, downregulating VEGF expression and inhibiting vascular-like formation on RF/6A.

Preparation Procedure and Pharmacokinetic Study of Water-in-Oil Nanoemulsion of Panax Notoginseng Saponins for Improving the Oral Bioavailability by Zhidong Liu, Qian Zhang, Lingling Ding, Chunhua Li, Zhongpeng Yin, Guoqiang Yan, Jiaxin Pi, Jiawei Li, Nan Li, Dongli Qi (600-610).
Background: Due to the effects of gastric acid, glycosidase and intestinal flora in the gastrointestinal environment, panax notoginseng saponins (PNS) can easily be resolved and metabolized when it is administered orally, limiting its oral bioavailability.
Methods: The formula of PNS nanoemulsion (PNS-N) was optimized using a pseudoternary phase diagram, and the PNS-N was prepared by high pressure homogenization. The type, particle size, polydispersity index (PDI), refractive index, pH and content of PNS-N were characterized. In vitro characteristics were investigated by drug release and physical stability. The pharmacokinetic properties of PNS-N were studied with rat intestine and SD rats. The optimized nanoemulsion formulation was Labrafil M 1944CS (58%), SP/EtOH (Km=1) (25%), solution of PNS (400mg/ml) (17%).
Results: The results showed that the average particle size was (28.17±0.39) nm with PDI of 0.116±0.032, refractive index of 1.4491±0.0009 and pH of 4.58±0.03. In addition, the contents of R1, Rg1 and Rb1 were (4.64±0.21) mg/mL, (19.16±0.27) mg/mL and (11.77±0.08) mg/mL, respectively. The optimized PNS-N formulation exhibited a sustained drug release with good stability. PNS-N is still clear and transparent, without layering and precipitation after six months. In the study of absorption kinetics of PNS-N in rat intestine, the Papp of three main components of PNS-N increased 5 times than PNS solution (PNS-SOL) in rat intestine. And pharmacokinetic study in SD rats suggested a 2.58-fold increase of oral bioavailability compared with PNS-SOL.
Conclusion: The PNS-N has increased the absolute availability of PNS obviously and nanoemulsion is a potential formulation to improve oral bioavailability for PNS.

Novel Thioester Prodrug of N-acetylcysteine for Odor Masking and Bioavailability Enhancement by Neha V. Bhilare, Suneela S. Dhaneshwar, Akanksha J. Sinha, Amit D. Kandhare, Subhash L. Bodhankar (611-620).
Background: The mucolytic N-acetylcysteine (NAC) is used to control the excessive mucus secretion if mucus is the underlying cause of broncho-constriction. Its major drawbacks are poor bioavailability due to extensive first pass effect, poor lipophilicity, high protein binding and offensive odor.
Methods: For minimizing above shortcomings of NAC, in present study thioester (A1) prodrug of NAC was synthesized by conventional as well as microwave-assisted methods. Release studies of A-1 were carried out using HPLC and pharmacological evaluation was performed in ovalbumin-induced model of pulmonary inflammation in Sprague dawley rats.
Results: A-1 was found to be stable in HCl buffer, phosphate buffer, stomach homogenates but furnished 30% NAC in 6h and 1.7% of NAC in 4h when incubated with small intestinal and liver homogenates respectively. Upon oral administration of A-1 to rats, 4.85% NAC was detected in blood at 8h. Urine samples pooled over a period of 24h exhibited 0.75% NAC while negligible concentration was found in 24 h pooled samples of feces.
Conclusion: The findings of this preliminary investigation demonstrated significant effects of thioester prodrug A-1 as compared to NAC through reduction of lung inflammation, airway eosinophilia and reversal of lung function parameters in ovalbumin- challenged rats at half the equimolar dose of NAC. Interestingly masking thiol group through thioester formation resulted in odorless prodrug. We propose that thioester prodrug using palmitic acid as a carrier is a promising strategy to enhance bioavailability of NAC by increasing its lipophilicity/ absorption and minimizing its first pass metabolism.

Background: Hydrogels are the polymeric network, which can retain large amount of water. Thus, these delivery systems always remained an issue of intensive research among the scientist fraternity.
Objective: In this piece of work, we have reconnoitered the significance of the PEGylated anticancer loaded drug onto poloxamer based thermoresponsive injectable hydrogel to understand the role of delivery system.
Method: To accomplish the objective, firstly it was necessary to improve the solubility of the melphalan, achieved by PEGylation of the drug with two grades of linear methoxy poly ethylene glycol (M-PEG), viz. M-PEG 2000 and 5000 Da to form a PEGylated melphalan conjugate (MLPEG). In our previous study, we have found that the prepared conjugates were efficiently enhanced the solubility of the melphalan and significantly reduced the hemolytic effect due to the presence of the PEG chains. Thus, in the present work, the prepared conjugates (MLPEG 5000 and 2000) were loaded to the thermosensitive Poloxamer 407 (P407) gel to produce an injectable hydrogel (MPX). To underline the reduction of the initial burst of the drug at the site of action, one of the hydrogels was prepared in the presence of the NaCl salt.
Results: This in turn, tightened the PEO chains and remarkably reduced the drug's initial burst from the delivery system as only 43 % of drug was released during 2 hours from MPX-CG hydrogel. Moreover, a lower diffusion coefficient (D) was noticed for MPX-CG gel as compared with MPX-7.4 gel. To confirm the depot formation, prepared hydrogels were administered to Wistar rats via subcutaneous and intramuscular routes.
Conclusion: Thus, P407 based injectable hydrogel could play an important role for the delivery of a low dose-alkylating agent with reduced host cytotoxicity.