A New Method for Targeted Drug Delivery Using Polymeric Microcapsules: Implications for Treatment of Crohn's Disease

A New Method for Targeted Drug Delivery Using Polymeric Microcapsules: Implications for Treatment of Crohn's Disease
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   A New Method for Targeted Drug Delivery Using Polymeric Microcapsules Implications for Treatment of Crohn’s Disease  Terrence Metz, 1  Mitchell L. Jones, 1 Hongmei Chen, 1 Trisnawati Halim, 1  Maryam Mirzaei, 1 Tasima Haque, 1 Devendra Amre, 2  Sujata K. Das, 3 and Satya Prakash*   ,1 1 Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Physiology, Artificial Cells & Organs Research Center, Faculty of Medicine, McGill University, 3775 University Street, Montreal, Quebec, H3A 2B4, Canada; 2  Sainte-Justine Hospital, University of Montreal, 3175, Cote-Sainte-Catherine, Montreal, Quebec, H3T 1C5, Canada; and 3 Division of Endocrinology, Department of Obstetrics and Gynecology, The N.Y. Hospital, Cornell University Medical Center, New York, NY 10021 Abstract Recent research and clinical evidence suggest that thalidomide could potentially be used to treatinflammation associated with Crohn’s disease. However, systemic side effects associated withlarge doses of this drug have limited its widespread use. Treatment with thalidomide would provemore efficacious if the drug could be delivered directly to target areas in the gut, thereby reducingsystemic circulation. Microcapsule encapsulation could enable direct delivery of the drug. Toassess the latter, we designed and tested drug-targeting release characteristics of alginate-poly- L -lysine-alginate (APA) microcapsules in simulated gastrointestinal environments. The results showthat APAcapsules enabled delivery of thalidomide in the middle and distal portions of the smallintestine. We also compared the APAmembrane formulation with an earlier designed alginate chi-tosan (AC) membrane thalidomide formulation. The results show that both APAand AC capsulesallow for successful delivery of thalidomide in the gut and could prove beneficial in the treatmentof Crohn’s disease. However, further research is required. Index Entries: Thalidomide; Crohn’s disease; artificial cells; microcapsule; inflammatory boweldisease. *Author to whom all correspondence and reprint requests should be addressed. E-mail: satya.prakash@ mcgill.ca O RIGINAL   A RTICLE  ©Copyright 2005 by Humana Press Inc.All rights of any nature whatsoever reserved.1085-9195/05/43:077–085/$30.00 Cell Biochemistry and Biophysics   77 Volume 43, 2005  INTRODUCTION Crohn’s disease is an inflammatory boweldisease (IBD) that causes inflammation in thegastrointestinal (GI) system. Adequate treat-ment of the disease has been hampered by theinability to determine specific factors involvedin perpetuating and maintaining the associatedinflammation. Nevertheless, based on evidenceimplicating the tumor necrosis factor (TNF)- α secretion pathway in Crohn-related inflamma-tion (1–5), current treatment modalitiesfocused on anti-TNF- α therapy have achievedsome measure of success in inducing andmaintaining remission of inflammatoryprocesses (6–8). Among these therapiesthalidomide has shown promise. Evidencefrom open-label clinical trials and other caseseries suggests that systemic administration of the drug could prove effective in providingpain relief, reducing the formation of ulcers,and stopping intestinal bleeding (8–15). However, well-known teratogenetic and neu-ropathic effects of the drug could limit its useover long periods of time, and long-term use isa prerequisite for maintaining remission andpreventing relapse (16–19). The initial promise shown by thalidomidecould be enhanced if methods to limit systemicadministration and simultaneous targeting toinflamed areas in the gut could be designed.Such methods could substantially augment theclinical efficacy of the drug and reduce associ-ated side effects. We have proposed that a ther-apy designed to orally deliver thalidomidewithin an artificial cell membrane engineered todegrade at a certain site along the GI pathwould overcome the limitations previouslyassociated with systemic delivery of thalido-mide. Encapsulation therapy would maintainthe drug within the microcapsule through thestomach, protecting it from being absorbed intothe systemic circulation. Artificial cell microen-capsulation is a method used for various appli-cations such as live cell implantation and drugdelivery (20–22). Earlier in vitro studies showedthat alginate chitosan (AC) membrane formula-tions have a burst-type thalidomide releasefrom the capsules after transfer from low to highpH that has potential for applications in IBD (23). This encourages researchers to develop anew formulation that can possibly have thecapacity to target other therapy areas of theintestine. The objective of the present study wasto design an alginate-poly- L -lysine-alginate(APA) membrane thalidomide formulation andevaluate its thalidomide release characteristicsfor targeted delivery of thalidomide. MATERIALS AND METHODS Chemicals and Equipment  Thalidomide (mol wt = 258.2), alginic acid(mol wt = 240,000; low viscosity), poly- L -lysine(hydrobromide) (mol wt [vis] = 27,400), anddimethyl sulfoxide (mol wt = 78.13) were pur-chased from Sigma-Aldrich (Ontario, Canada).Sodium citrate (mol wt = 294.10) was pur-chased from Fisher (Ontario, Canada).Chitosan 10 (low viscosity) was purchasedfrom Wako (Richmond, VA) and used for allAC microcapsule preparations. AnEncapsulator Research IER-20 pump (InotechBiosystems Rockville, MD) was used forencapsulation, a Varian Cary 100 BioSpectrophotometer (Varian, Palo Alto, CA) wasused for spectrophotometric studies, and aLab-Line Environ Shaker 3527 (Lab-LineDesigners and Manufacturers, Melrose Park, IL60160-1491, USAwere used for incubationstudies. Preparation of APA Thalidomide  Microcapsule Formulation  Alginic acid (Sigma) was added to deionizedwater to make a 1.5% alginate solution. (–)-Thali-domide ([–]-2-[2,6-dioxo-3-piperidinyl]-1H-iso-indole-1,3[2H]-dione) (Sigma) was dissolved indeionized water at a concentration of 0.035mg/mLby stirring and heating for 24 h andadded to the alginate solution. Alginic acid wasadditionally added to maintain a 1.5% concen-tration after the thalidomide and water solutionwas included. APAbeads were then formed by 78Metz et al. Cell Biochemistry and BiophysicsVolume 43, 2005  running the aforementioned solution through anInotech encapsulator pump using a 300- µ m noz-zle. The frequency was set to 528 Hz, the flowrate to 20.8 mL/min, and the voltage to 0.348 kV.Formed beads were collected in a prepared 0.1  M calcium chloride solution to avoid cell aggrega-tion. The beads were then washed with deion-ized water and soaked in a 0.1% poly- L -lysine(Sigma) bath for 10 min. The beads were washedagain and soaked in 0.15% alginate solution for15 min. Afinal washing was done with water,and the beads were transferred into calciumchloride for storage. The capsules were visuallyevaluated for uniformity and integrity through aLomo light microscope with × 250 magnification.  Measurement of Efficacy of Thalidomide  Encapsulation in APA Microcapsule Formulation  Thalidomide encapsulation was evaluatedinitially through APAmembrane degradation.Samples of beads weighing 35 mg weresoaked in a prepared solution of 3% sodiumcitrate for 12 h in order to dissolve the cellmembrane. Samples from these preparationswere analyzed in a Varian Cary 100 UV-visiblespectrophotometer (Varian Canada, St. Laurent,Quebec) for thalidomide detection at a 220-nmwavelength and compared with the super-natant of the beads prior to soaking in sodiumcitrate. Testing of APA Microcapsule Thalidomide Formulation’s Stability and Thalidomide  Release in Simulated GI Fluid  Samples of APA(1.22 and 1.30 g dry wt) beads containing thalidomide were washed,filtered, and added to a prepared pH 1.5 buffersolution for 10 min to simulate acidic condi-tions normally encountered in the stomach.The solutions were shaken at 125 rpm in anEnviron shaker. The microcapsules were thentransferred to a pH 7.5 buffer solution andshaken to simulate proximal small intestineconditions for 60 min. For the duration of bothtests, supernatant samples were spectrophoto-metrically analyzed every 10 min. Testing of APA Microcapsule Thalidomide Formulation’s Stability in Variable pH  Environments Asuitable amount of APAmicrocapsuleswas counted and put in 5-cm Petri dishes with10 mLof pH 1.5 physiological buffer solutions.Measured quantities of pH 7.4 physiological buffer solution were added every 5 min inorder to increase gradually the pH of each indi-vidual dish to values of 1.2., 2.0, 2.6, 3.18, 4.77,5.64, 6.15, 6.5, 6.93, and 7.4, respectively. Aftereach addition of pH 7.4 buffer, the capsules ineach dish were counted and observed fordegradation. RESULTS We studied the feasibility of deliveringthalidomide to the proximal and middle smallintestine, where Crohn’s disease-related inflam-mation most commonly occurs. Experimentswere designed to formulate APAcapsules con-taining thalidomide and analyze the drug’srelease in simulated GI pH conditions. Figure 1shows the APAmicrocapsules containingthalidomide. The results show that APAbeads Polymeric Microcapsules for Drug Delivery79  Cell Biochemistry and BiophysicsVolume 43, 2005 Fig. 1.Photomicrograph of APAmicrocap-sule thalidomide formulation under × 250magnification.  containing thalidomide were regular in forma-tion with diameters measuring 300–350 µ m (Fig.1). Additional experiments were designed toconfirm thalidomide retention by the APAmembrane. We challenged the APAthalidomideformulation to a sodium citrate solution for 12 hand analyzed the presence of thalidomide in thesupernatant solutions using a spectrophotome-ter. The results show the presence of a high con-centration of thalidomide in the solution.Alternatively, in control studies, analysis of membrane supernatant prior to sodium citrateexposure showed no thalidomide.Microcapsule stability in various pH condi-tions was studied. The results show that theAPAmembrane was stable from pH 1.2 to 5.8.However, membrane thinning was not detectedto a great extent until pH values reached arange of 6.15–6.5 (Fig. 2). Furthermore, whentested after 10 min of shaking in the pH 1.5 buffer solution, thalidomide release from APAmicrocapsules was minimal. The release of thalidomide from the APAcapsules acts as atimed mechanism slowly allowing the drug toescape as the membrane degrades. Full releaseof thalidomide was achieved after 60 min of shaking. Thalidomide peaks remained constantat an absorbance of 1.6 after 60 min indicated bythe highest peak detected (Fig. 3).APAmembrane degradation was studiedand the results show that the APAmembranehad a thinning effect (Fig. 4). Studies were alsodesigned to compare APAmicrocapsules withan earlier proposed AC microcapsule formula-tion to evaluate their stability and delivery (24) (Fig. 5). Differences in degradation of the twomembranes in the pH range of 4.77–6.15 seemmost likely to be owed to differences in ionic bonding between the two-layer AC membraneand the three-layer APAmembrane. The three-layer ionic bonding of the APAcapsule mem- brane could lead to the thinning results, ratherthan immediate degradation, as seen with theAC capsules.The results show that both of these mem- branes delivered the drug to separate areas of the gut through differing degradation patterns.AC microcapsules when exposed to pH 7.5 for20 min, revealed a full degradation of the mem- brane. Alternatively, in the APAmembrane for-mulation, the alginate layer degraded first,leaving the polylysine membrane layer intact,more slowly while allowing the thalidomide to be released. These degradation differences 80Metz et al. Cell Biochemistry and BiophysicsVolume 43, 2005 Fig. 2.Comparative study of APAcapsule degradation in a pH-varying environment.   between the APAand AC microcapsules at var-ious pH conditions are shown in Fig. 5. AC cap-sule degradation, as reported above, occurredin a burstlike fashion. Specifically between 80and 100% of the capsules were intact through-out the test until pH levels rose above 3.18.Between pH 3.18 and 5.64, nearly 70% of the ACcapsules had burst. Conversely, APAcapsulesthinned out as the alginate coatings degraded,producing a ghosting effect (Fig. 4). DISCUSSION Previous studies have evaluated treatmentwith thalidomide for oral, vulvar, distal intesti-nal (jejunum and ileum), and colonic localizedCrohn’s disease (25–29). However, the fullpotential of thalidomide has not been realized because of its associated side effects. It has beensuggested that targeted delivery could be idealfor thalidomide therapy. We have shown thatencapsulation could be an alternative methodfor thalidomide delivery. We observed the dif-ferent mechanisms of drug delivery from bothAPAand AC membranes (Fig. 6). Although theliterature suggests that the molecular mem- brane permeability of AC and APAmembranesis much larger than the thalidomide molecule,the fact that these membranes can retain thedrug is a subject for further investigation. We,however, hypothesized that the drug was main-tained within the capsules owing to bindingforces between the negatively charged alginate Polymeric Microcapsules for Drug Delivery81 Cell Biochemistry and BiophysicsVolume 43, 2005 Fig. 3.Thalidomide release from APAmicrocapsule formulation in simulated GI pH of 7.5.Fig. 4.Photomicrograph of APAmem- branes after exposure to pH 7.5 conditions for20 min ( × 250 magnification).
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