The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`Development and Characterization of Biodegradable Chitosan Films for Local
`Delivery of Paclitaxel
`Submitted: January 23, 2004; Accepted: June 22, 2004; Published: October 11, 2004.
`Anand Babu Dhanikula,1 and Ramesh Panchagnula1
`1Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar
`160062, India
`
`ABSTRACT
`Intratumoral and local drug delivery strategies have gained
`momentum recently as a promising modality in cancer ther-
`apy. In order to deliver paclitaxel at the tumor site in thera-
`peutically relevant concentrations, chitosan films were fabri-
`cated. Paclitaxel could be loaded at 31% wt/wt in films,
`which were translucent and flexible. Physicochemical char-
`acterization of paclitaxel via thermal, spectroscopic, x-ray
`diffraction, and electron microscopy techniques revealed
`information on solid-state properties of paclitaxel as well as
`chitosan in films. While chitosan was in amorphous form,
`paclitaxel seemed to be present in both amorphous and crys-
`talline forms in film. The polymeric dispersion of paclitaxel
`in poloxamer formed fibrous structures generating disconti-
`nuities in the film matrix, thereby leading to the introduction
`of perturbations in the packing arrangement of polymer
`chains. These films released only 10% to 15% of loaded
`paclitaxel by a burst effect under in vitro testing conditions,
`with lysozyme having no effect on the release. However,
`films softened after implantation in mice and lost integrity
`over time. The implantable delivery system is not only
`biodegradable but also well tolerated in vivo and hence, bio-
`compatible as revealed by histological studies. The lack of
`formulation-induced local inflammatory responses of pacli-
`taxel chitosan films suggests a new paradigm for localized
`chemotherapy based on implantable systems.
`
`KEYWORDS: paclitaxel, local delivery, film, solid-state,
`histology, mice.
`
`INTRODUCTION
`In recent years, biodegradable polymeric systems have gained
`importance for design of surgical devices, artificial organs,
`drug delivery systems with different routes of administration,
`carriers of immobilized enzymes and cells, biosensors, ocular
`Corresponding Author: Ramesh Panchagnula, Department
`of Pharmaceutics, National Institute of Pharmaceutical,
`Education and Research (NIPER), Sector 67, Phase X, SAS
`Nagar, 160062 (Punjab) India. Tel: 91-172-2214682,
`2214687. Fax: 91-172-2214692. Email: panchagnula@
`yahoo.com.
`
`inserts, and materials for orthopedic applications.1 These
`polymers are classified as either synthetic (polyesters,
`polyamides, polyanhydrides) and natural (polyamino acids,
`polysaccharides).2 Polysaccharide-based polymers represent
`a major class of biomaterials, which includes agarose, algi-
`nate, carageenan, dextran, and chitosan.
`Chitosan, β(1,4)2-amino-2-D-glucose, is a cationic biopoly-
`mer produced by alkaline N-deacetylation of chitin, which is
`the main component of the shells of crab, shrimp, and krill.
`Chitosan has found many biomedical applications, including
`tissue engineering, owing to its biocompatibility, low toxici-
`ty, and degradation in the body by enzymes such as chi-
`tosanase and lysozyme,3 which has opened up avenues for
`modulating drug release in vivo in the treatment of various
`diseases. These chitosan-based delivery systems range from
`microparticles to nanoparticles4 to gels5 and films.6 Further,
`gels and films of chitosan have been used for oral delivery of
`chlorhexidine digluconate in the treatment of fungal infec-
`tions.7 In addition, chitosan has been extensively evaluated
`as a carrier of various antineoplastic agents such as 5-fluo-
`rouracil,8 mitoxantrone,9 cytarabine,10 and paclitaxel.11
`The film-forming property of chitosan has found many appli-
`cations in tissue engineering and drug delivery by virtue of
`its mechanical strength and rather slow biodegradation.12
`Some drug-loaded chitosan films are emerging as novel drug
`delivery systems,13-14 and films appear to have potential for
`local sustained delivery of cancer chemotherapeutic agents.
`Following surgical removal of tumor, these implantable sys-
`tems may be placed in the resection cavity to elicit a local
`response at the biophase; further, they may be secured by
`suturing at the site to prevent any displacement problems.
`Though paclitaxel is the most extensively investigated anti-
`cancer drug in the last 3 decades, it is not a good option for
`the treatment of brain tumors after systemic administration.15
`Successful treatment of malignant brain tumors is alarmingly
`negligible because antineoplastic agents, including paclitaxel,
`have limited access to the tumor site across the blood brain
`barrier when administered systemically. An alternative
`approach to systemic delivery of antineoplastic drugs is local-
`ized delivery from a polymer matrix. In the field of local
`delivery, carmustine-loaded Gliadel wafer (Guilford
`Pharmaceuticals, Baltimore, MD) fabricated from poly(car-
`boxyphenoxy propane:sebacic acid) proved very promising in
`
`1
`
`Page 1
`
`Mylan v. MonoSol
`IPR2017-00200
`MonoSol Ex. 2022
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`and dried at 60°C for 12 hours. After preparation, all films
`clinical trials for the treatment of malignant glioma, increas-
`were stored in airtight containers for further studies.
`ing both survival and safety.16 The objective of this study was
`to develop a chitosan film-based local delivery system for
`sustained release of paclitaxel to tumor site after implantation.
`These films have been evaluated for the release of impregnat-
`ed paclitaxel, characterized by physical techniques and
`microscopy, and examined for inflammatory reactions by his-
`tological examination after implantation in mice.
`
`MATERIALS AND METHODS
`Materials
`Paclitaxel was a gift sample from Dabur India Ltd (Uttar
`Pradesh, India). Radioactive paclitaxel (14C) of specific
`activity 42.5 mCi/mmol, chitosan (≥85% deacetylated),
`lysozyme, and Tween 80 were purchased from Sigma (St
`Louis, MO). Poloxamer 407 was obtained as gratis sample
`from BASF (Ludwigshafen, Germany). Soya phosphatidyl-
`choline and phosphatidylglycerol were kindly provided by
`Nattermann & Cie Gmbh (Cologne, Germany). Absolute
`ethanol (ETOH) was procured from Merck KgaA
`(Darmstadt, Germany). Glycerol and glacial acetic acid were
`obtained from LOBA Chemie (Mumbai, India). High pres-
`sure liquid chromatography (HPLC)-grade methanol was
`obtained from (J.T. Baker, Madero, Mexico). Thiopentone
`sodium and gentamicin were of parenteral grade. All other
`reagents were analytical or reagent grade. Water obtained
`from ELGA purification unit (Marlow, UK) was used
`throughout the study. All animal experimentation was per-
`formed in accordance with protocols approved by the institu-
`tional animal ethical committee.
`
`Preparation of Paclitaxel Chitosan Films
`Because paclitaxel is a hydrophobic drug, 2 different
`approaches were used for its incorporation into chitosan
`films: one involved only phospholipids and the other used
`poloxamer 407 in presence of ETOH. Initially, a 10 mg/mL
`chitosan solution was prepared in 1% (vol/vol) acetic acid,
`and glycerol was included as a plasticizer at a chitosan:glyc-
`erol weight ratio of 2:1. In the first method, liposomes con-
`taining paclitaxel (spiked with radioactive component) were
`prepared by film hydration method using phosphatidyl
`choline and phosphatidyl glycerol (9:1, soya origin) at 6 to
`12 mol% drug loading and were subsequently dispersed in
`chitosan solution. Then, film was cast by pouring the mixture
`on a glass plate (area 45.5 cm2) followed by drying under
`vacuum for 48 hours at 37°C. In the second method, required
`quantities of paclitaxel and poloxamer 407 were dissolved
`separately in 1 mL of ETOH and mixed together, and the
`ethanolic solution was added to chitosan solution and agitat-
`ed to disperse paclitaxel. Subsequently, the homogeneous
`suspension was cast into film by the method described above
`
`Stability of Paclitaxel During Film Preparation
`The following procedure was used to assess the stability of
`paclitaxel during the film preparation process. The prepared
`films were extracted twice with a solvent mixture of 1:1 ace-
`tonitrile and ETOH (vol/vol); the extract was evaporated; the
`residue obtained was reconstituted in mobile phase; and an
`aliquot was injected onto HPLC column. Stability-indicating
`chromatographic method was adopted for this purpose
`(Waters Corp, Milford, MA).17 The method consisted of a
`Symmetry C18 column (250 × 4.6 mm; 5 µm) run using a
`mobile phase of composition methanol:water (70:30 vol/vol)
`at a flow rate of 0.5 mL/min, a Waters pump (600 E), and elu-
`ants monitored with Waters photodiode array detector (996
`PDA) at 227 nm.
`
`Content Uniformity of Films
`To ensure uniform distribution of paclitaxel in film, a content
`uniformity test was performed. Samples representing differ-
`ent regions within film were cut and weighed, and paclitaxel
`was extracted with a 1:1 solvent mixture of acetonitrile and
`ETOH (vol/vol) twice for 12 hours each time at room tem-
`perature. These extracts were pooled for liquid scintillation
`counting (EG&G Wallac, Turku, Finland).
`
`Release Studies
`A definite weight range of 10-15mg of film was cut and
`placed in a 1.5-mL capacity microcentrifuge tube containing
`1 mL of release medium of the following composition at
`37°C: phosphate buffered saline (140 mM, pH 7.4) with
`0.1% sodium azide and 0.1% Tween 80. At predetermined
`time points, 100 µL of release medium was sampled with
`replacement to which 3 mL of scintillation cocktail was
`added and vortexed before liquid scintillation counting. The
`cumulative amount of paclitaxel released as a function of
`time was calculated. In addition, to simulate the in vivo con-
`ditions, release of paclitaxel in presence of lysozyme (2
`mg/100 mL) was also studied.
`
`Film Thickness
`Film thickness was measured using a micrometer (Mitutoyo,
`Kanagawa, Japan) with the smallest possible unit measure-
`ment count of 0.01 mm.
`
`Tensile Strength
`The effect of paclitaxel on mechanical properties of chitosan
`films was assessed through a tensile strength test. Tensile
`
`2
`
`Page 2
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`ination (Jeol Fine Coat, ion sputter, JFC-1100). The speci-
`strength of film was measured using texture analyzer TA-
`XT2i (Stable Micro Systems, Surrey, UK) with the following
`mens were scanned with an electron beam of 1.2 kV acceler-
`acquisition parameters:
`ation potential, and images were collected in secondary elec-
`tron mode.
`1. 2 mm/s prespeed
`2. 1 mm/s test-speed
`3. 10 mm/s postspeed with an acquisition rate of 50
`points/s
`4. 5 kg load cell
`Film was secured with tensile grips, and a trigger force of 5 g
`was applied. The resulting profiles were analyzed using Texture
`Expert, Version 1.22 (Stable Micro Systems, Surrey, UK).
`
`X-ray Diffraction Studies
`Molecular arrangement of paclitaxel and chitosan in powder
`as well as in films was compared by powder x-ray diffraction
`patterns acquired at room temperature on a Philips PW 1729
`diffractometer (Eindhoven, Netherlands) using Cu Kα radia-
`tion. The data were collected over an angular range from 3°
`to 50° 2θ in continuous mode using a step size of 0.02° 2θ
`and step time of 5 seconds.
`
`Solid-State Characterization
`To study the molecular properties of paclitaxel and chitosan,
`the solid-state characterization was done by the application
`of thermal, infrared, x-ray diffraction, and microscopy tech-
`niques. During these studies, solid-state characteristics of
`paclitaxel and chitosan were compared with those of film to
`reveal any changes occurring as a result of film preparation.
`
`Differential Scanning Calorimetry
`Differential scanning calorimetry (DSC) studies were per-
`thermal analyzer
`formed with a Mettler Toledo 821e
`(Greifensee, Switzerland) calibrated with indium as standard.
`For thermogram acquisition, sample sizes of 1 to 5 mg were
`scanned with a heating rate of 5°C/min over a temperature
`range of 25°C to 300°C. In order to check the reversibility of
`transition, samples were heated to a point just above the cor-
`responding transition temperature, cooled to room tempera-
`ture, and reheated up to 300°C.
`
`Fourier Transform Infrared Spectroscopy
`Fourier transform infrared (FTIR) spectra were obtained for
`paclitaxel, chitosan, blank films, and paclitaxel films on
`Nicolet Impact 410 (Nicolet Analytical Instruments,
`Madison, WI). Spectra of paclitaxel and chitosan were
`obtained using the potassium bromide disc method, while
`those of films were acquired directly. In each case, 100 spec-
`tra in the region of 400 to 4000 cm-1 were co-added with a
`resolution of 2 cm-1.
`
`Scanning Electron Microscopy
`Paclitaxel samples and chitosan films were viewed using a
`Jeol scanning electron microscope (SEM), JSM 1600
`(Tokyo, Japan) for morphological examination. Powder sam-
`ples of paclitaxel and films were mounted onto aluminum
`stubs using double-sided adhesive tape and then sputter coat-
`ed with a thin layer of gold at 10 Torr vacuum before exam-
`
`In Vivo Implantation Studies
`Biodegradation of films was studied in Swiss mice. Initially,
`mice were anesthetized with thiopentone sodium (40 mg/kg)
`and occasional light ether inhalation, and an incision was
`made in the back of the neck region with a scalpel. After inci-
`sion, the implantation site was created by tunneling immedi-
`ately beneath the skin, then films were inserted and the skin
`was sutured. To prevent infection, mice were given gentam-
`icin (2 mg/kg, intraperitoneal route) every 4 days. For in vivo
`implantation purposes, film was prepared in a plastic mold of
`radius 1.15 cm (instead of glass plate) with each mouse
`receiving one such film.
`
`Histology Studies
`Histology studies were performed to examine the acute toxi-
`city of film at the implantation site. After a 2-month implan-
`tation period, mice were humanely killed by cervical disloca-
`tion and an incision was made in the implantation area. Then,
`the tissue in which the film was imbibed was removed and
`stored in 50% formalin until processing. Subsequently, tissue
`processing involved dehydration through a graded series of
`alcohols (70%, 80%, 95%, and 100%), followed by xylene
`and then infiltration with paraffin. For obtaining thin sections
`(3-5 µm), tissues were embedded on the edge of paraffin
`blocks and were cut on a rotary microtome. These sections
`were deparafinized, rehydrated with graded alcohols (100%,
`95%, 80%, and 75%), and stained with hemotoxylin/eosin
`for microscopic examination.18 Similarly, sections of pacli-
`taxel chitosan film and tissue of healthy mouse were
`obtained to serve as control.
`
`RESULTS
`Preparation of Films
`The difficulty of incorporating water-insoluble paclitaxel mol-
`ecules was circumvented by concomitant inclusion of unsatu-
`
`3
`
`Page 3
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`
`Table 1. Composition and Thickness of Paclitaxel Chitosan Films Obtained by Casting Method*
`
`Film
`Poloxamer 407
`Phosphatidyl
`Phosphatidyl
`Thickness
`(µm)
`(mg)§
`Glycerol (mg)‡
`Choline (mg)‡
`Paclitaxel (mg)
`Chitosan (mg)† Glycerol (mg)
`Film Code
`50-60
`10
`-
`15
`130
`200
`100
`FLM-1-PCL
`50-60
`15
`-
`15
`130
`200
`100
`FLM-2-PCL
`50-60
`20
`-
`15
`130
`200
`100
`FLM-3-PCL
`40-45
`10
`50
`-
`-
`150
`100
`FLM-4-PCL
`100-120
`20
`10
`-
`-
`25
`10
`FLM-5-PCL||
`*FLM indicates film. Films 1, 2, 3, and 4 were cast on glass plate of surface area 45.5 cm2, while film 5 was cast in plastic mold of surface area
`4.2 cm2. Films 1, 2, and 3 were dried at 37°C; films 4 and 5 were dried at 60°C.
`†Chitosan is ≥85% deacetylated.
`‡Liposomes containing paclitaxel were prepared from phospholipids of soya origin by film hydration method and used without extrusion; encapsula-
`tion efficiency ~95%.
`§The type of poloxamer used here could be replaced with other grades or other polymers which might lead to better formulation.
`||Formulation was selected for physicochemical characterization and histology studies (see text).
`method. For this purpose, paclitaxel was extracted from film
`rated phospholipids (in the form of liposomes) or poloxamer
`along with paclitaxel in film (Table 1). Unsaturated lipids were
`and analyzed by HPLC. A single peak at 18.5 minutes repre-
`used to prepare liposomes (as a means of incorporation of
`senting paclitaxel (with no additional peaks) was detected in
`paclitaxel in film) since earlier studies have shown that these
`the chromatogram, suggesting that the molecule was stable
`lipids yield best encapsulation efficiency of paclitaxel (D.A.
`during preparation of films (chromatograms not shown).
`and R.P., unpublished data, 2000). These films were obtained
`by casting method and appeared either transparent or translu-
`cent and were pale yellowish in color. Further, poloxamer-con-
`taining films showed good loading capacities of 20 mg of
`paclitaxel per 25 mg of chitosan, and these films had a mean
`weight of 63.5 mg. At this loading percentage (30%-31%),
`essentially all drug could be incorporated into film without any
`precipitation. A lower fabrication temperature of 37°C was
`chosen for lipid films in contrast to poloxamer films (60°C) in
`order to minimize hydrolysis and oxidation of unsaturated
`phospholipids. When films were examined for thickness, the
`lipid films (FLM-1,2,3-PCL) ranged from 50 to 60 µm, while
`poloxamer films FLM-4-PCL (cast on glass plate) ranged from
`40 to 45 µm, and FLM-5-PCL (cast in plastic mold) ranged
`from 100 to 120 µm. At constant casting surface area, the high-
`er thickness of lipid films (FLM-1,2,3-PCL) over poloxamer
`films (FLM-4-PCL) is due to phospholipids and a higher
`amount of chitosan. Although initial attempts to incorporate
`paclitaxel with phospholipids were found to be feasible, since
`unsaturated lipids are prone to oxidation, only chitosan-polox-
`amer films containing paclitaxel were chosen for further char-
`acterization unless specified.
`
`Content Uniformity
`Paclitaxel was extracted from different regions of chitosan
`film using acetonitrile:ETOH (1:1 vol/vol) solvent system.
`After normalization of amount of paclitaxel on weight basis
`of film, the results indicated that the variation in distribution
`of paclitaxel in different regions of film was <15% (results
`not shown).
`
`Release Studies
`In order to establish the ability of films to serve as depot for-
`mulations, release of paclitaxel from both lipid- and polox-
`amer-containing films was studied. It was observed that
`release was negligible from lipid-containing films, while
`those containing poloxamer showed a burst effect followed
`by no release. Films containing poloxamer released ~10% in
`6 hours; further release of paclitaxel was not observed until
`the study period of 144 hours, suggesting that the film had
`retained 90% of the payload (Figure 1A). Since the release of
`paclitaxel was <10%, further studies were undertaken in
`presence of lysozyme (chitosan is a substrate for the enzyme)
`to simulate the in vivo conditions. However, no significant
`difference was observed in presence of lysozyme (Figure
`1B), suggesting that this model was not appropriate to simu-
`late in vivo conditions for release-rate studies.
`
`Mechanical Strength of Film
`Mechanical strength of film is described in terms of tensile
`strength, and brittle films are characterized by a decrease in
`
`4
`
`Chemical Stability of Paclitaxel
`In the present study, films were prepared by the classical
`method, which involves spreading a uniform layer of polymer
`dispersion followed by a drying step for removal of solvent
`system. Since film preparation methodology involved a heat-
`ing step, it may have had a detrimental effect on the chemical
`stability of drug. Hence, stability assessment of paclitaxel
`impregnated in film was done using stability-indicating
`
`Page 4
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`
`Figure 1. Cumulative percentage release (in vitro) of
`paclitaxel from chitosan film at 37°C in (A) absence and
`(B) presence of lysozyme (2 mg/100mL); 0.1% Tween 80
`was used in release medium to provide sink conditions
`(data are mean ± SD; n = 3).
`
`Figure 2. Force-time profiles of (A) blank film and (B)
`paclitaxel chitosan film to study the influence of formula-
`tion approach on mechanical properties of film.
`the percentage of elongation at break. The area under curve
`is related to the energy required to break polymeric material,
`and tough polymers have larger areas requiring large
`amounts of energy for rupture. In order to understand the
`arrangement of polymer chains in the presence of paclitaxel,
`force-time profiles of films were generated as shown in
`Figure 2. Although force of elongation at break is slightly
`lowered in presence of paclitaxel in film (10.3 vs 9.8 N), the
`area under the profile has been increased (31.3 vs 45.9 N).
`For convenience of interpretation, each profile is further
`described in terms of ascending and descending portions.
`The time to plateau of the ascending portion of paclitaxel-
`chitosan film was greater in comparison with control film. As
`brittleness is reflected in the time to plateau, greater time is
`
`5
`
`Figure 3. DSC studies to investigate physical transforma-
`tions induced in chitosan and paclitaxel by comparing
`solid-state features of pure components with that in films
`(A) paclitaxel, (B) chitosan powder, (C) blank film, and
`(D) paclitaxel chitosan film. Thermograms were obtained
`at a scan rate of 5°C/min. When paclitaxel chitosan film
`was heated to 190°C, cooled to 25°C, and reheated, peaks
`I, II, and III were found to be irreversible in nature.
`indicative of lack of brittleness of film. In addition, the
`descending segment of the profile of control film was uni-
`form, while that of paclitaxel film was irregular and protract-
`ed. The discontinuities in internal structure and variation in
`strength of film matrix may be the cause of the irregular
`descending portion of the profile.
`
`Solid-state Characterization
`Thermal Studies of Films
`The DSC thermograms of paclitaxel, recrystallized paclitaxel,
`blank, and paclitaxel-chitosan films are shown in Figure 3,
`and the observed thermal events are summarized in Table 2.
`Thermogram of paclitaxel showed an initial broad peak at
`64.5°C (Peak I, Figure 3A) due to removal of absorbed mois-
`ture or nonstructural water followed by a single endotherm at
`223.6°C (Peak III, Figure 3A) just prior to an exotherm of
`degradation peak. Another minor broad peak at 168.9°C
`(Peak II, Figure 3A) was observed, which is due to the pres-
`ence of small amounts of paclitaxel dihydrate in the sample19
`(the peak was absent from second heating phase on DSC run
`when the sample was initially heated to 200°C, cooled back
`to 25°C, and reheated; results not shown). Liggins et al19 have
`previously ascribed this peak to solid-solid transition associ-
`ated with the conversion of dehydrated paclitaxel dihydrate to
`semicrystalline form. DSC studies were also performed on
`dry powder (recrystallized paclitaxel) obtained by evapora-
`
`Page 5
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`
`Table 2. Details of Thermal Events Obtained by Differential Scanning Calorimetry for Paclitaxel, Recrystallized Paclitaxel,
`Chitosan Powder, Blank Chitosan Film, and Paclitaxel Chitosan Film*
`Sample
`Peak I
`Peak II
`
`Peak V
`Peak IV
`Peak III
`∆H
`∆H
`∆H
`∆H
`T
`T
`T
`T
`T
`241.1
`-
`-
`54.69
`223.6
`17.17
`168.9
`22.24
`64.5
`Paclitaxel
`219.6
`2.19
`197.4
`5.81
`63.2
`6.29
`49.7
`4.54
`38.1
`Recrystallized paclitaxel
`294.5
`-
`-
`-
`-
`-
`-
`145.63
`68.2
`Chitosan powder
`274.5
`129.22
`230.4
`0.72
`139.9
`78.99
`85.3
`6.93
`48.3
`FLM-5-BLK
`280.1
`36.65
`230.9
`15.28
`172.5
`92.65
`88.5
`9.46
`48.1
`FLM-5-PCL
`*T indicates peak temperature in °C; and ∆H, enthalpy of peak in J/g. Peaks I, II, III, and IV are endothermic, while peak V represents exothermic
`events. Peaks I, II, III, and IV are due to transitions/melting, while peak V is a result of decomposition. Peak number of one sample has no relevance
`with peak number of other samples. Peaks I, II, and III are irreversible events for film samples. Decomposition peak of chitosan was considerably
`broadened on casting into film. Note that although SEM photomicrographs of recrystallized paclitaxel and paclitaxel film reveal the presence of
`microparticles, the corresponding melting endotherms were not observed here, probably owing to lack of sensitivity of DSC to detect the minute per-
`centage of the same in samples.
`tion of paclitaxel-poloxamer mixtures dispersed in aqueous
`acetic acid-ETOH solvent system (with same proportions as
`mentioned in Table 1). The only differences were that chi-
`tosan and glycerol were omitted. In DSC, thermograms of
`recrystallized paclitaxel (not shown), a single endotherm of
`transition of poloxamer at 49.7°C (Peak II, Table 2), and 2
`minor transitions were observed. However, a transition peak
`of paclitaxel with very low heat of fusion was seen at 197.4°C
`(Peak IV, Table 2), while the melting endotherm at 223.6°C
`was absent. On the other hand, the degradation peak of pacli-
`taxel appeared at 219.6°C (Peak V, Table 2).
`Both blank and paclitaxel-chitosan films exhibited 4
`endothermic peaks in DSC thermograms. The transition of
`poloxamer in these films occurred at 48°C (Peak I, Figures 3C
`and D) with no appreciable shift. Other peaks in the region of
`85°C to 88°C (Peak II) have resulted from loss of moisture on
`heating. In paclitaxel-loaded film, an asymmetric peak repre-
`senting thermal event of a new form of paclitaxel was seen at
`172.5°C (Peak III, Figure 3D). This event was followed by a
`broad endothermic peak at 230°C to 240°C (Peak V, Figures
`3C and D) in both films, which may be attributed to glycerol
`component. However, thermal events of paclitaxel, namely,
`melting and decomposition, which were previously noted in
`the region of 190°C to 225°C were not observable in film
`(Figure 3D). Further, decomposition of chitosan was observed
`as a broad exotherm in films at 274°C to 280°C, while the
`same decomposition exotherm was at 294.5°C (Peak V,
`Figure 3B) for chitosan powder under identical experimental
`DSC conditions.
`
`3431 cm-1, which is assigned to the N-H and hydrogen bond-
`ed O-H stretch vibrational frequencies, while a sharp (shoul-
`der) peak at 3610 cm-1 is that of free O-H bond stretch of glu-
`copyranose units. Further, in the C-H stretch region of FTIR
`spectrum, the higher intensity peak at 2923 cm-1 is assigned
`to the asymmetric and the lower intensity peak at 2857 cm-1
`is assigned to the symmetric modes of CH2. In addition, the
`characteristic band due to CH2 scissoring, which usually
`occurs at 1465 cm-1 was also present in the sample. Since the
`grade of chitosan used in the present study was ≥85%
`deacetylated, an amide bond peak was present in the spectra
`and the C=O stretch of amide bond was observed at 1661
`cm-1. The peaks at 1550 and 1599 cm-1 were assigned to
`strong N-H bending vibrations of secondary amide, which
`usually occur in the range of 1640 to 1550 cm-1 as strong
`band.20
`In comparison to the chitosan powder, spectrum was not
`sharp in film. An overlay of chitosan film and paclitaxel-chi-
`tosan film is shown in Figures 4B and C. The presence of
`residual moisture content and glycerol in films resulted in a
`broad peak from 3500 to 2800 cm-1. The peak at 1651 cm-1,
`representative of C=O stretch of amide bond in chitosan film,
`shifted to 1648 cm-1 in the presence of paclitaxel. Further, the
`peak due to N-H bending vibration, which was observed at
`1588.6 cm-1 in chitosan film, was lowered to 1579 cm-1 in
`paclitaxel-chitosan film; its intensity also decreased. The
`band due to CH2 scissoring, which occurred at 1466 cm-1,
`was broadened due to paclitaxel in film. However, only 2
`characteristic peaks of paclitaxel, at 1740 and 1706 cm-1,
`were observed in paclitaxel-chitosan film, while other peaks
`were not discernible due to interference caused by polymers.
`In order to abstract spectral features of paclitaxel, infrared
`spectra of control chitosan film were subtracted from that of
`paclitaxel-chitosan film as shown in Figure 4E. A clear loss
`in resolution of infrared features of paclitaxel in film was
`observed when compared with that of powder paclitaxel
`(Figure 4D). The peak due to C=O amide stretch at 1646 cm-1
`
`Fourier Transform Infrared Spectroscopy
`Transmission infrared spectra of chitosan powder, paclitaxel,
`and films were acquired to draw information on the molecu-
`lar state of chitosan and paclitaxel (Figure 4A). Chitosan is
`an amino glucose characterized by a small proportion of
`amide groups via an amide linkage with acetic acid. In the
`infrared spectrum, powder chitosan exhibited a broad peak at
`
`6
`
`Page 6
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`
`Figure 4. FTIR spectra of (A) chitosan powder, (B) blank film, (C) paclitaxel chitosan film, (D) paclitaxel, and (E) subtrac-
`tion result of blank film from paclitaxel chitosan film to examine the alterations in molecular vibrations of paclitaxel and
`chitosan induced as a result of film fabrication. All spectra are plotted in transmittance mode.
`B. In addition, SEM photomicrographs of control and paclitax-
`considerably broadened in film in comparison with powder
`el films were acquired and compared with that of paclitaxel
`paclitaxel. Further, the peak representing CH2 scissoring
`samples. The morphology of control film was plain and the pic-
`mode of paclitaxel at 1451 cm-1 decreased in intensity in
`ture appeared dark, indicating that the control film has a smooth
`film. A shift in wave number owing to CH3 bending at 1370
`surface (Figure 5C). In contrast to control film, features of
`cm-1 of paclitaxel to 1366 cm-1 occurred.
`paclitaxel-chitosan film resembled that of fibrous recrystallized
`paclitaxel morphology (Figure 5D). This typical surface
`appearance suggests that paclitaxel is not only dispersed in the
`film matrix but also projected onto the surface of film. Some
`irregularly shaped particles were also identified in the pictures
`of recrystallized paclitaxel and film loaded with paclitaxel.
`
`Film Morphology Studies
`SEM photomicrographs revealed that commercial sample of
`paclitaxel has a plate-like appearance, while recrystallized sam-
`ple appear fibrous and elongated, as shown in Figures 5A and
`
`7
`
`Page 7
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`
`Figure 5. Scanning electron photomicrographs of (A) paclitaxel, (B) recrystallized paclitaxel, (C) blank film, and (D)
`paclitaxel chitosan film obtained at different magnifications. Paclitaxel photomicrographs were that of commercial sample
`without any modification. Recrystallized paclitaxel was obtained by evaporation of hydro-ethanolic solution of paclitaxel
`and poloxamer 407 under identical conditions as used for film casting in the absence of chitosan (see Table 1).
`Magnification: (A) × 5000 (left); × 15 000 (right); (B) × 10 000 (left); × 15 000 (right); (C) × 5000 (left); × 20 000 (right);
`(D) × 5000 (left); × 15 000 (right). The arrows in (D) indicate the presence of poloxamer coated paclitaxel microparticles.
`
`X-ray Diffraction Studies
`X-ray diffraction is a proven tool to study crystal lattice
`arrangements and yields very useful information on degree
`of sample crystallinity. X-ray diffraction pattern of pacli-
`taxel, blank, and paclitaxel film were obtained and com-
`pared, which revealed marked differences in the molecular
`state of paclitaxel (Figure 6). The diffractogram of blank
`chitosan film has shown 2 low intensity peaks at 19.1° and
`23.3° 2θ with a characteristic broad hump in the range of 7°
`to 45° 2θ. This halo diffraction pattern (broad hump) is an
`indication of the predominantly amorphous form of chi-
`tosan in films (Figure 6A). In the case of paclitaxel, the dif-
`fractogram exhibited peaks at the following 2θ values: 4.1°,
`5.4°, 5.8°, 9.1°, 10.2°, 11.3°, 12.3°, and 12.5° (Figure 6B).
`Among these, the peak of highest intensity was located at
`5.8° 2θ, and the peaks at 11.3° and 12.5° 2θ were broad.
`When the diffraction pattern of paclitaxel in chitosan film
`was compared with that of paclitaxel, the pattern differed to
`a large extent. Several high-angle diffraction peaks were
`observed in paclitaxel-chitosan film at the following 2θ val-
`ues: 4.1°, 5.2°, 6.2°, 10.8°, 12.1°, 12.6°, 13.8°, 14.6°, 18.6°,
`20°, 20.7°, 21.6°, and 23.6° (Figure 6C). The 12.6° 2θ peak
`had the highest intensity, and the hump in the baseline
`occurred from 7° to 45° 2θ, as observed for chitosan film.
`
`Figure 6. X-ray diffraction patterns of (A) blank chitosan
`film, (B) paclitaxel, and (C) paclitaxel chitosan film at
`ambient temperature to examine solid-state features of
`paclitaxel in film (the overlay picture is obtained by trac-
`ing out individual patterns by hand).
`
`8
`
`Page 8
`
`

`

`The AAPS Journal 2004; 6 (3) Article 27 (http://www.aapsj.org).
`surrounding tissues, and after 50 days it was c