Polysialic acid as a drug carrier: evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers

This paper explores the potential of polysialic acid (PSA) as a carrier for low molecular weight anticancer drugs. A PSA–epirubicin (Epi) conjugate was synthesized and compared against Epi conjugates containing established carriers, namely: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, poly(ethylene glycol) (PEG) and polyglutamic acid (PGA). Biological assessments in the breast cancer cell line MCF-7 and in the anthracycline resistant MCF-7/DX showed that the PSA–Epi conjugate had the highest activity (40% and 30% cell death in the two cell lines at 1 mM Epi equiv., respectively). FACS studies confirmed internalization of all conjugates by cholesterol-dependent endocytosis. PSA–Epi showed release of Epi (40% at 5 h) when incubated with lysosome extracts. In vivo evaluation showed that all conjugates had a significantly longer half-life compared to free Epi. This study also allowed an investigation on the effect of the polymeric carrier on the biological activity of a conjugate, with the biodegradability of the carrier emerging as an important feature.

Study of quercetin-loaded liposomes as potential drug carriers: in vitro evaluation of human complement activation

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC: CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC: CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.

Niosomes as Nanoparticular Drug Carriers: Fundamentals and Recent Applications

Drug delivery systems are defined as formulations aiming for transportation of a drug to the desired area of action within the body. The basic component of drug delivery systems is an appropriate carrier that protects the drug from rapid degradation or clearance and thereby enhances drug concentration in target tissues. Based on their biodegradable, biocompatible, and nonimmunogenic structure, niosomes are promising drug carriers that are formed by self-association of nonionic surfactants and cholesterol in an aqueous phase. In recent years, numerous research articles have been published in scientific journals reporting the potential of niosomes to serve as a carrier for the delivery of different types of drugs. The present review describes preparation methods, characterization techniques, and recent studies on niosomal drug delivery systems and also gives up to date information regarding recent applications of niosomes in drug delivery.

Liposomes and micro/nanoparticles as colloidal carriers for nasal drug delivery

The use of the nasal route for drug delivery has attracted much interest in recent years in the pharmaceutical field. Local and principally systemic drug delivery can be achieved by this route of administration. But the nasal route of delivery is not applicable to all drugs. Polar drugs and some macromolecules are not absorbed in sufficient concentration due to poor membrane permeability, rapid clearance and enzymatic degradation into the nasal cavity. Thus, alternative means that help overcome these nasal barriers are currently in development. Absorption enhancers such as phospholipids and surfactants are constantly used, but care must be taken in relation to their concentration. Drug delivery systems including liposomes, cyclodextrins, micro- and nanoparticles are being investigated to increase the bioavailability of drugs delivered intranasally. This review article discusses recent progress and specific development issues relating to colloidal drug delivery systems in nasal drug delivery. © 2006 Bentham Science Publishers Ltd.

A biodegradable drug delivery system for the treatment of postoperative inflammation

Cataract surgery is often performed in patients suffering from associated pathologies. Our goal is to develop a biodegradable drug delivery system (DDS) combined with the artificial intraocular lens (IOL). DDS were manufactured using poly(D,L-lactide-co-glycolide), or PLGA, and were loaded with triamcinolone acetonide (TA). The loading capacity was approximately 1050 microg of TA per DDS. The higher the molecular weight of PLGA (34,000, 48,000 and 80,000Da), the slower was the release of TA in vitro. Cataract surgery was performed on the right eye of rabbits. IOL was inserted with (i) no DDS, (ii) unloaded DDS PLGA48000, (iii) one loaded DDS PLGA48000, (iv) two loaded DDS. The number of inflammatory cells and the protein concentration were measured in the aqueous humor (AH). Unloaded DDS showed good ocular biocompatibility. One DDS PLGA48000 loaded with TA significantly reduced postoperative ocular inflammation. Two loaded DDS PLGA48000 was even more effective in inhibiting such inflammation. On long-term observation (days 63 and 84), reduction of inflammation could be obtained by insertion of one DDS PLGA48000 and a second DDS PLGA80000. Therefore, our "all in one" system is very promising since it could replace oral treatment and reduce the number of intraocular injections

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Effect of polymer architecture on Curcumin 1 encapsulation and release from PEGylated polymer nanoparticles: toward a drug delivery nano-platform to the CNS

We developed a nanoparticles (NPs) library from poly(ethylene glycol)–poly lactic acid comb-like polymers with variable amount of PEG. Curcumin was encapsulated in the NPs with a view to develop a delivery platform to treat diseases involving oxidative stress affecting the CNS. We observed a sharp decrease in size between 15 and 20% w/w of PEG which corresponds to a transition from a large solid particle structure to a “micelle-like” or “polymer nano-aggregate” structure. Drug loading, loading efficacy and release kinetics were determined. The diffusion coefficients of curcumin in NPs were determined using a mathematical modeling. The higher diffusion was observed for solid particles compared to “polymer nano-aggregate” particles. NPs did not present any significant toxicity when tested in vitro on a neuronal cell line. Moreover, the ability of NPs carrying curcumin to prevent oxidative stress was evidenced and linked to polymer architecture and NPs organization. Our study showed the intimate relationship between the polymer architecture and the biophysical properties of the resulting NPs and sheds light on new approaches to design efficient NP-based drug carriers.

Can drug-bearing liposomes penetrate intact skin?

Using liposomes to deliver drugs to and through human skin is controversial, as their function varies with type and composition. Thus they may act as drug carriers controlling release of the medicinal agent. Alternatively, they may provide a localized depot in the skin so minimizing systemic effects or can be used for targeting delivery to skin appendages (hair follicles and sweat glands). Liposomes may also enhance transdermal drug delivery, increasing systemic drug concentrations. With such a multiplicity of functions, it is not surprising that mechanisms of liposomal delivery of therapeutic agents to and through the skin are unclear. Accordingly, this article provides an overview of the modes and mechanisms of action of different vesicles as drug delivery vectors in human skin. Our conclusion is that vesicles, depending on the composition and method of preparation, can vary with respect to size, lamellarity, charge, membrane fluidity or elasticity and drug entrapment. This variability allows for multiple functions ranging from local to transdermal effects. Application to dissimilar skins (animal or human) via diverse protocols may reveal different mechanisms of action with possible vesicle skin penetration reaching different depths, from surface assimilation to (rarely) the viable tissue and subsequent systemic absorption.

Xyloglucan nano-aggregates: Physico-chemical characterisation in buffer solution and potential application as a carrier for camptothecin, an anti-cancer drug

In this work, native xyloglucan was extracted from Tamarindus indica seeds (XGT), and its properties in phosphate buffer solution (PBS) were evaluated in comparison with a commercial tamarind kernel powder (TKP). The physico-chemical characteristics of the polysaccharides such as molar mass, critical concentration and intrinsic viscosity were determined. Furthermore, using spectroscopic and microscopy techniques, it was observed that the XGs tested can be considered macromolecules able to aggregate as nano-entities of 60-140 nm. The XGT tended to an ordered and compact spherical conformation determined by the Huggins constant, circular dichroism, atomic force microscopy and transmission electron microscopy. After the determination of the properties in PBS the XGs, at concentrations of 25% above their critical aggregation concentration, were used to encapsulate camptothecin, an anti-cancer drug. The XGT sample showed an encapsulation efficiency of 42% and first-order drug delivery kinetics. These results demonstrated the importance of knowledge of the physico-chemical properties of polysaccharides, for example, to better conduct their biotechnological applications as drug carriers. (C) 2010 Elsevier Ltd. All rights reserved.

Development and in vitro evaluation of coated pellets containing chitosan to potential colonic drug delivery

In this work pellets containing chitosan for colonic drug delivery were developed. The influence of the polysaccharide in the pellets was evaluated by swelling, drug dissolution and intestinal permeation studies. Drug-loaded pellets containing chitosan as swellable polymer were coated with an inner layer of Kollicoat® SR 30 D and an outer layer of the enteric polymer Kollicoat® MAE 30 DP in a fluidized-bed apparatus. Metronidazole released from pellets was assessed using Bio-Dis dissolution method. Swelling, drug release and intestinal permeation were dependent on the chitosan and the coating composition. The drug release data fitted well with the Weibull equation, indicating that the drug release was controlled by diffusion, polymer relaxation and erosion occurring simultaneously. The film coating was found to be the main factor controlling the drug release and the chitosan controlling the drug intestinal permeation. Coated pellets containing chitosan show great potential as a system for drug delivery to the colon. © 2012 Elsevier Ltd.

Cyclodextrin Inclusion Complexes Loaded in Particles as Drug Carrier Systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of drug physicochemical properties on the release from liposomal systems in vitro and in vivo

Liposomes were discovered about 40 years ago by A. Bangham and since then they became very versatile tools in biology, biochemistry and medicine. Liposomes are the smallest artificial vesicles of spherical shape that can be produced from natural untoxic phospholipids and cholesterol. Liposome vesicles can be used as drug carriers and become loaded with a great variety of molecules, such as small drug molecules, proteins, nucleotides and even plasmids. Due to the variability of liposomal compositions they can be used for a large number of applications. In this thesis the β-adrenoceptor antagonists propranolol, metoprolol, atenolol and pindolol, glucose, 18F-Fluorodeoxyglucose (FDG) and Er-DTPA were used for encapsulation in liposomes, characterization and in vitro release studies. Multilamellar vesicles (MLV), large unilamellar vesicles (LUV) and smaller unilamellar vesicles (SUV) were prepared using one of the following lipids: 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), Phospholipone 90H (Ph90H) or a mixture of DSPC and DMPC (1:1). The freeze thawing method was used for preparation of liposomes because it has three advantages (1) avoiding the use of chloroform, which is used in other methods and causes toxicity (2) it is a simple method and (3) it gives high entrapping efficiency. The percentage of entrapping efficiencies (EE) was different depending on the type and phase transition temperature (Tc) of the lipid used. The average particle size and particle size distribution of the prepared liposomes were determined using both dynamic light scattering (DLS) and laser diffraction analyzer (LDA). The average particle size of the prepared liposomes differs according to both liposomal type and lipid type. Dispersion and dialysis techniques were used for the study of the in vitro release of β-adrenoceptor antagonists. The in vitro release rate of β-adrenoceptor antagonists was increased from MLV to LUV to SUV. Regarding the lipid type, β-adrenoceptor antagonists exhibited different in vitro release pattern from one lipid to another. Two different concentrations (50 and 100mg/ml) of Ph90H were used for studying the effect of lipid concentration on the in vitro release of β-adrenoceptor antagonists. It was found that liposomes made from 50 mg/ml Ph90H exhibited higher release rates than liposomes made at 100 mg/ml Ph90H. Also glucose was encapsulated in MLV, LUV and SUV using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), Phospholipone 90H (Ph90H), soybean lipid (Syb) or a mixture of DSPC and DMPC (1:1). The average particle size and size distribution were determined using laser diffraction analysis. It was found that both EE and average particle size differ depending on both lipid and liposomal types. The in vitro release of glucose from different types of liposomes was performed using a dispersion method. It was found that the in vitro release of glucose from different liposomes is dependent on the lipid type. 18F-FDG was encapsulated in MLV 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), Phospholipone 90H (Ph90H), soybean lipid (Syb) or a mixture of DSPC and DMPC (1:1). FDG-containing LUV and SUV were prepared using Ph90H lipid. The in vitro release of FDG from the different types of lipids was accomplished using a dispersion method. Results similar to that of glucose release were obtained. In vivo imaging of FDG in both uncapsulated FDG and FDG-containing MLV was performed in the brain and the whole body of rats using PET scanner. It was found that the release of FDG from FDG-containing MLV was sustained. In vitro-In vivo correlation was studied using the in vitro release data of FDG from liposomes and in vivo absorption data of FDG from injected liposomes using microPET. Erbium, which is a lanthanide metal, was used as a chelate with DTPA for encapsulation in SUV liposomes for the indirect radiation therapy of cancer. The liposomes were prepared using three different concentrations of soybean lipid (30, 50 and 70 mg/ml). The stability of Er-DTPA SUV liposomes was carried out by storage of the prepared liposomes at three different temperatures (4, 25 and 37 °C). It was found that the release of Er-DTPA complex is temperature dependent, the higher the temperature, the higher the release. There was an inverse relationship between the release of the Er-DTPA complex and the concentration of lipid.

Tissue engineering strategies for biomaterial-based anticancer drug delivery and enhancement of angiogenesis in tumor-resected bone

Patienten, die an Osteosarkom leiden werden derzeit mit intravenös applizierten krebstherapeutischen Mitteln nach Tumorresektion behandelt, was oftmals mit schweren Nebenwirkungen und einem verzögerten Knochenheilungsprozess einhergeht. Darüber hinaus treten vermehrt Rezidive aufgrund von verbleibenden neoplastischen Zellen an der Tumorresektionsstelle auf. Erfolgreiche Knochenregeneration und die Kontrolle von den im Gewebe verbleibenden Krebszellen stellt eine Herausforderung für das Tissue Engineering nach Knochenverlust durch Tumorentfernung dar. In dieser Hinsicht scheint der Einsatz von Hydroxyapatit als Knochenersatzmaterial in Kombination mit Cyclodextrin als Medikamententräger, vielversprechend. Chemotherapeutika können an Biomaterial gebunden und direkt am Tumorbett über einen längeren Zeitraum freigesetzt werden, um verbliebene neoplastische Zellen zu eliminieren. Lokal applizierte Chemotherapie hat diverse Vorteile, einschließlich der direkten zytotoxischen Auswirkung auf lokale Zellen, sowie die Reduzierung schwerer Nebenwirkungen. Diese Studie wurde durchgeführt, um die Funktionsfähigkeit eines solchen Arzneimittelabgabesystems zu bewerten und um Strategien im Bereich des Tissue Engineerings zu entwickeln, die den Knochenheilungsprozess und im speziellen die Vaskularisierung fördern sollen. Die Ergebnisse zeigen, dass nicht nur Krebszellen von der chemotherapeutischen Behandlung betroffen sind. Primäre Endothelzellen wie zum Beispiel HUVEC zeigten eine hohe Sensibilität Cisplatin und Doxorubicin gegenüber. Beide Medikamente lösten in HUVEC ein tumor-unterdrückendes Signal durch die Hochregulation von p53 und p21 aus. Zudem scheint Hypoxie einen krebstherapeutischen Einfluss zu haben, da die Behandlung sensitiver HUVEC mit Hypoxie die Zellen vor Zytotoxizität schützte. Der chemo-protektive Effekt schien deutlich weniger auf Krebszelllinien zu wirken. Diese Resultate könnten eine mögliche chemotherapeutische Strategie darstellen, um den Effekt eines zielgerichteten Medikamenteneinsatzes auf Krebszellen zu verbessern unter gleichzeitiger Schonung gesunder Zellen. Eine erfolgreiche Integration eines Systems, das Arzneimittel abgibt, kombiniert mit einem Biomaterial zur Stabilisierung und Regeneration, könnte gesunden Endothelzellen die Möglichkeit bieten zu proliferieren und Blutgefäße zu bilden, während verbleibende Krebszellen eliminiert werden. Da der Prozess der Knochengeweberemodellierung mit einer starken Beeinträchtigung der Lebensqualität des Patienten einhergeht, ist die Beschleunigung des postoperativen Heilungsprozesses eines der Ziele des Tissue Engineerings. Die Bildung von Blutgefäßen ist unabdingbar für eine erfolgreiche Integration eines Knochentransplantats in das Gewebe. Daher ist ein umfangreich ausgebildetes Blutgefäßsystem für einen verbesserten Heilungsprozess während der klinischen Anwendung wünschenswert. Frühere Experimente zeigen, dass sich die Anwendung von Ko-Kulturen aus humanen primären Osteoblasten (pOB) und humanen outgrowth endothelial cells (OEC) im Hinblick auf die Bildung stabiler gefäßähnlicher Strukturen in vitro, die auch effizient in das mikrovaskuläre System in vivo integriert werden konnten, als erfolgreich erweisen. Dieser Ansatz könnte genutzt werden, um prä-vaskularisierte Konstrukte herzustellen, die den Knochenheilungsprozess nach der Implantation fördern. Zusätzlich repräsentiert das Ko-Kultursystem ein exzellentes in vitro Model, um Faktoren, welche stark in den Prozess der Knochenheilung und Angiogenese eingebunden sind, zu identifizieren und zu analysieren. Es ist bekannt, dass Makrophagen eine maßgebliche Rolle in der inflammatorisch-induzierten Angiogenese spielen. In diesem Zusammenhang hebt diese Studie den positiven Einfluss THP-1 abgeleiteter Makrophagen in Ko-Kultur mit pOB und OEC hervor. Die Ergebnisse zeigten, dass die Anwendung von Makrophagen als inflammatorischer Stimulus im bereits etablierten Ko-Kultursystem zu einer pro-angiogenen Aktivierung der OEC führte, was in einer signifikant erhöhten Bildung blutgefäßähnlicher Strukturen in vitro resultierte. Außerdem zeigte die Analyse von Faktoren, die in der durch Entzündung hervorgerufenen Angiogenese eine wichtige Rolle spielen, eine deutliche Hochregulation von VEGF, inflammatorischer Zytokine und Adhäsionsmoleküle, die letztlich zu einer verstärkten Vaskularisierung beitragen. Diese Resultate werden dem Einfluss von Makrophagen zugeschrieben und könnten zukünftig im Tissue Engineering eingesetzt werden, um den Heilungsprozess zu beschleunigen und damit die klinische Situation von Patienten zu verbessern. Darüber hinaus könnte die Kombination der auf Ko-Kulturen basierenden Ansätze für das Knochen Tissue Engineering mit einem biomaterial-basierenden Arzneimittelabgabesystem zum klinischen Einsatz kommen, der die Eliminierung verbliebener Krebszellen mit der Förderung der Knochenregeneration verbindet.