Received 7 January 2014, Revised 31 March 2014, Accepted 1 April 2014 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3201
Topical delivery of fluorescence (6-Cf) labeledand radiolabeled (99m-Tc) cisplatin andimiquimod by a dual drug delivery systemVandana Gupta,a* Krishna Chuttani,b Anil K. Mishra,b and Piyush Trivedia
The present investigation deals with the development of topica
l (top.) formulation for co-delivery of cisplatin and imiquimod toenhance the antitumor efficacy of the drug for skin-cited malignancies even in immune compromised patient. Cisplatin (CDDP)and imiquimod-loaded protransfersome gel (CDDP-Imi-Pts gel) formulation was characterized for entrapment efficiency, pH,and viscosity. Further, fluorescence-labeled (6-carboxyfluorescin) and radiolabeled (99mtechnetium) drug-loaded formulationswere compared with respect to biodistribution and pharmacokinetic studies. Gamma scintigraphy of mice following radiolabeledformulation administrationswas performed to accomplish the localization of drugs in various organs. The percentage entrapmentefficiency of cisplatin and imiquimod in the protransfersome gel formulations were found to be 36.22±6.41 and 63.11±3.73.The skin/blood localization ratio of 1.096, 120.13, 0.174, and 349.88 was found for intraperitoneal radiolabeled drug solution(99m-Tc-CDDP-Imi-Sol), top. radiolabeled drug-loadedprotransfersomegel formulation (99m-Tc-CDDP-Imi-Pts gel), intraperitoneal6-carboxyfluorescin labeled drug solution (6-Cf-CDDP-Imi-Sol), top. 6-carboxyfluorescin labeled drug-loaded protransfersomegel formulation (6-Cf-CDDP-Imi-Pts gel), respectively after 0.5h of administration. CDDP-Imi-Pts gel has a potential for sitespecific delivery and reduces the systemic toxicity of anti cancer drugs. These findings suggest that the cisplatin–imiquimodco-delivery offers an attractive, novel approach for treatment of skin-cited malignancies.
aSchool of Pharmaceutical Sciences, Rajiv Gandhi Technical University, GandhiNagar, Airport Bypass Road, Bhopal 462033, India
bDivision of Cyclotron & Radiopharmaceutical Sciences, Institute of NuclearMedicine and Allied Sciences (INMAS), DRDO, Delhi 110054, India
*Correspondence to: Vandana Gupta, School of Pharmaceutical Sciences, RajivGandhi Technical University, Gandhi Nagar, Airport Bypass Road, Bhopal462033, India.E-mail: [email protected]
42
Introduction
Cutaneous malignancy is by far the most common of allpathologies related to cancerous disease.1 It constitutes one ofthe most common human malignancies. Annually, more than400,000 people find out that they have skin cancer. Etiology isrelated to various factors such as skin type, age, sun exposure,and many other factors. Predisposing factors include individualswith a fair or light complexion, a history of severe sunburns, poortanning capability, inherited disorders (e.g., xeroderma pigmentosa,and albinism), and immunocompromise.2 In addition cutaneouscarcinomas are the most frequent cancers in organ transplantrecipients.3
Non-melanoma skin cancers (NMSC) are the common humancancers and most NMSC develop on sun exposed areas of thebody. UV radiation is the most important risk factor forpathogenesis of basal cell and squamous cell carcinoma, the effecton risk of squamous cell carcinoma is the greatest.4,5
Topical imiquimod is an ‘immune response facilitator’ that appearsto effectively treat both viral disorders (human papillomavirusinfection and molluscum contagiosum) and some cutaneousmalignancies (actinic keratosis, squamous cell carcinoma in situ,and superficial basal cell carcinoma) by augmenting both innateand acquired immune response.6,7
Cisplatin (CDDP), cis-diaminedichloro-platinum (II), is one ofthe first line chemotherapeutic agents used in the treatment ofcancer. The cytotoxic effects of cisplatin are mediated by itsinteraction with DNA, resulting in the formation of DNA adductswhich activate several signal transduction pathways and
J. Label Compd. Radiopharm 2014, 57 425–433
culminate in the activation of apoptosis. However, use ofcisplatin in cancer chemotherapy is limited because of acquiredor intrinsic resistance of cells to the drug.8,9
To reduce cancer drug resistance for better therapeuticeffectiveness, combination chemotherapy has long beenadopted in clinics as a primary cancer treatment. Co-delivery ofdrugs, targeting the same cellular pathways could functionsynergistically for higher therapeutic efficacy and higher targetselectivity. Further, combination therapy with two or moreanticancer drugs with same drug delivery system is thecornerstone of cancer therapy.10
Over the past decade, topical delivery of drugs has gainedmore and more attention of which systemic side effects can bereduced compared with parenteral or oral drug administration.Moreover, combination therapy often proves more efficaciousand better tolerated than monotherapy with a single drug.11,12
An array of topical drug therapies is available for the treatmentof NMSC, the potential of various delivery strategies has been
investigated to modulate vesicle composition in order to improvethe ability of nanocarriers to carry drugs and macromolecules todeeper tissues, leading to two novel vesicular carriers, ultraflexiblelipid-based elastic vesicles (transfersomes) and ethosomes thatgave the most promising results. Ultradeformable liposomes haveshown potential as a carrier for topical drug delivery systemsbecause they can penetrate the skin intact.13 Ultraflexible vesicles,transfersomes are the biphasic first generation of elastic vesiclesintroduced and were reported to penetrate intact skin consistingof phospholipids and an edge activator,14,15 which destabilizelipid bilayers and increase deformability of the vesicles as wellas accommodate both drugs, that is, hydrophilic and lipophilicin a single delivery system.16 Proultraflexible lipid vesicles,protransfersomes (Pts) would be converted into ultraflexiblevesicles in situ by absorbing water from the skin.17
The objective of the present work was to prepare and evaluatecisplatin and imiquimod-loaded protransfersome gel (CDDP-Imi-Pts gel) formulation, which is an elastic lipid vesiclesembedded in carbopol gel network. It is proposed that CDDP-Imi-Pts gel formulation will provide site specific delivery of bothdrugs, that is, cisplatin and imiquimod and can reduceunwanted systemic toxicities. Furthermore, cisplatin deliverythrough topical delivery system in combination with imiquimodmay potentiate the activity against cutaneous malignancieseven in immune compromised patient.
Materials and methods
Drugs and reagents
Imiquimod was procured as a gift sample from GlanmarkPharmaceuticals Limited (Mumbai, India). Soya lecithin (phospha-tidylcholine) was supplied by ACROS ORGANICS (NJ, USA).Cisplatin, sodium cholate, pluronic F-68, and sodium diethyldithio-carbamate were procured from Sigma Chemical Co. (St. Louis, MO,USA). Carbopol 940 was purchased from Himedia (Mumbai, India).Reagents and organic solvents such as disodium hydrogenphosphate, potassium dihydrogen phosphate, sodium chloride,sodium hydroxide, and isopropanol were purchased from CDHPvt. Ltd. (New Delhi, India). All other reagents were of analyticalor HPLC grade, and the water used was double distilled, deionized,and filtered with water purification system (Younglin, Aqua Max,Ultra 370 Series).
Animals
Swiss albino mice (25–30 g) were selected for the biodistributionand pharmacokinetic studies. Permission for the use ofexperimental animals was obtained from institutional animalethical committees (INMAS, New Delhi, India). Three animals foreach formulation per time point were used in the studies.
Methods
Preparation of cisplatin and imiquimod-loaded protransfersomeformulation
Cisplatin and imiquimod-loaded protransfersome was preparedaccording to the reported method18 with minor modification.Briefly, Soya lecithin (1.7 g) and stabilizer (pluronic F-68, 0.15 g)were taken in a clean, dry wide mouth tube and mixed well sothat pluronic would be able to incorporate inside the lipid bilayer,followed by surfactant (sodium cholate, 0.15 g), imiquimod (0.05g)
and isopropanol (1.5ml), warmed in a water bath at 60–70 °C untilthe ingredients were dissolved.Cisplatin (0.01 g) in 2ml of phosphate buffer saline (PBS),
(pH 7.4) was added dropwise to the wide mouth tube andwarmed on a water bath until a clear solution was formed. Thismixture was kept cooling at room temperature for an hour.
Incorporation of CDDP-Imi-Pts formulation in carbopol gel base
For the preparation of CDDP-Imi-Pts gel, the best achievedCDDP-Imi-Pts system was incorporated into 2% (w/v) carbopolgel base. The specified amount of carbopol powder was slowlyadded to purified water and allowed to hydrate for 4–5 h. Thedispersion was neutralized with 10% (w/v) aqueous solution ofNaOH to pH 5.5. Eventually, a viscous solution was obtained.Appropriate amounts of CDDP-Imi-Pts system were incorporatedinto prepared carbopol gel base with continuous stirring untilhomogenous gel was achieved.
Determination of pH, viscosity, and percentages of drug contentand entrapment efficiency
The pH value of the CDDP-Imi-Pts gel formulation was determineddirectly in samples at room temperature by using pH tester (Eutechwaterproof pH Testr® 10 (Eutech Instrument technology, USA)).The DVII + Pro Brookfield viscometer (Brookfield Engineering
Laboratories, Stoughton, MA, USA, with software) with smallsample adaptor (spindal and chamber SC4-18/13R) was used todetermine flow properties of the CDDP-Imi-Pts gel formulationbetween the percentage of torque values of 10–100.Cisplatin and imiquimod-loaded protransfersome gel formulation
(100mg) was hydrated with 10ml of 0.9% saline (0.9% NaCl):methanol (8:2) and manually shaken for 5min. About 1ml ofthe resultant solution was taken and diluted with solvent up to10ml and then absorbance was recorded using a UV/Visiblespectrophotometer (Shimadzu 1700, Japan). Each experimentwas performed in triplicate. A placebo formulation preparedusing a process similar to that used for preparing the sampleswas also subjected to spectrophotometric measurements.The total drug content was calculated for each drug using thefollowing equation: TDC= concentration×dilution factor × volumeof formulation/total amount of drug added×100.The entrapment efficiency was determined after separating
the unentrapped drug. CDDP-Imi-Pts gel formulation (100mg)was hydrated with 10ml of 0.9% saline (0.9% NaCl): methanol(8:2) and manually shaken for 5min. After that, for the separationof the unentrapped drug, the sample was subjected tocentrifugation at 5000 rpm for 30min. The clear supernatantwas siphoned off carefully to separate the unentrapped drug.About 1ml of this supernatant was taken and diluted with10ml of solvent and the absorbance was recorded. The sediment(1ml) was resuspended in 1ml of triton x-100 (0.2% v/v). Afterappropriate dilution, the absorbance was recorded. The cisplatinand imiquimod entrapment efficiency was calculated by thefollowing equation: percentage entrapment = amount of drugin sediment/total amount of drug added× 100. Amount of drugin supernatant and sediment gave a total amount of drug.Simultaneous estimation of cisplatin and imiquimod wasperformed by dual wavelength method using UV/Visiblespectrophotometer (Shimadzu 1700). So for the estimation ofcisplatin, two wavelengths (220 and 258 nm) were selectedwhere imiquimod has a similar absorbance and imiquimod canbe estimated at its maxima (245 nm).
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V. Gupta et al.42
Permeation studies
Swiss albino mice skin of full thickness was taken for studies. Aftercleaning with cold tap water, full thickness, non-dermatome skinwas removed with the help of a scalpel. Locally fabricated Franzdiffusion cell was used for the skin permeation studies. Cells had aneffective diffusion area of 0.785cm2. The receptor compartment wasfilled with 10ml of PBS (pH7.4) and stirred with a magnetic bar. Thetemperature of the receptor compartment was maintained at37±1 °Cwith an external, constant temperature circulator water bath.The skin specimen was sandwiched between the donor and
the receptor compartments of the Franz diffusion cell with thestratum corneum facing the donor compartment. Formulations,cisplatin, and imiquimod solution (drug solution) in 8:2 ratio of0.9% NaCl: methanol (CDDP-Imi-Sol) and CDDP-Imi-Pts gelequivalent to 200μg of cisplatin and 1mg of imiquimod wereplaced in the donor compartment. At predetermined time intervals,samples (1.0ml) were taken from the receptor compartment andreplaced with an equivalent amount of fresh buffer solution tomaintain sink condition.The cumulative amount of cisplatin and imiquimod permeated
per cm2 of skin was calculated and plotted as a function of time.The permeation parameters were calculated from the linear partof the plot, which corresponds to the steady state, being the flux(J), the slope, and the lag time (Tlag). The permeability coefficient(Kp) was obtained dividing the flux by the drug concentration inthe donor compartment.
Preparation of radiolabeled drug formulations
Drug solution (CDDP-Imi-Sol) and gel formulation (CDDP-Imi-Ptsgel) were radiolabeled with technetium-99m (99m-Tc) by direct-labeling method.19,20 Stannous chloride dihydrate (100μg in100μl of 0.10 N HCl) was added each to 0.5ml of CDDP-Imi-Soland CDDP-Imi-Pts gel formulation. The pH was adjusted to 6.5using a 0.5M sodium bicarbonate solution. Sterile 99m-Tc-pertechnetate was added with continuous mixing, and themixture was incubated at room temperature for 10min. Theactivity of the resultant solution was 1.5mCi/ml.A radiochemical purity/stability of the optimized radiolabeled
formulations was determined by dialysis: a small volume of labeledformulation (0.5ml) in normal saline was placed in a dialysis sac(pore size 60 kD) and dialyzed against 100ml of normal saline at37 °C for 24h. Aliquots from the dialysates were taken and theradioactivity counted in triplicate. The radioactivity was alsodetermined before incubation.The radiolabelling efficiency was determined after separating
the unlabelled radioactive material. The formulations were stirredfor 15min at room temperature, and then centrifuged, followedby washing three times with saline to make it free from unlabelled99m-Tc. After washing, radioactivity present in 99m-Tc-CDDP-Imi-Ptsgel formulation was measured using shielded well-type gammascintillation counter (CAPRAC WELL COUNTER, CAPINTEC, CAPRAC-R,Southern Scientific Ltd, United Kingdom). Results of radiolabellingefficiency were calculated as the percentage of the total amountof radioactivity added by using the following equation: percentageradiolabelling= counts in formulation after washing/total countsadded×100.
Preparation of fluorescence-labeled drug formulations
The fluorescent labeling was carried out by preparing the CDDP-Imi-Sol and CDDP-Imi-Pts gel formulation with a fluorescenceprobe, 6-carboxyfluorescin (0.05%).
Biodistribution study forms an integral part of site specific drugdelivery and is necessary to show that the developedformulation is both effective and safe by avoiding nonspecificdistribution to other organs of the body thereby avoidingprospective drug related toxicity. Swiss albino mice weighing25–30 g who were kept on a synthetic pellet diet and water adlibitum were divided into four groups (groups 1–4) containing03 mice in each group. The back of the animals of groups 1and 3 was shaved 1 day prior to the start of the experiment.Group 1 being topically treated with 99m-Tc-CDDP-Imi-Pts gel,Group 2 with intraperitoneal (i.p.) 99m-Tc-CDDP-Imi-Sol, Group 3with topical 6-Cf-CDDP-Imi-Pts gel, and Group 4 was treated withi.p. 6-Cf-CDDP-Imi-Sol. Each animal was dosed with 200μl(containing 200μg of cisplatin and 1mg of imiquimod). Samplesof blood were collected from the animals at times 0.5, 1.0, 2.0,4.0, 6.0, 8.0, and 24 h post-administration by cardiac puncture.Subsequently, other tissues (lung, liver, spleen, kidney, and skin)were removed, washed twice using normal saline, made freefrom adhering tissue, and weighed. For the groups 1 and 2,radioactivity present in each tissue/organ was measured usingshielded well-type gamma scintillation counter (CAPRAC WELLCOUNTER, CAPINTEC, CAPRAC-R). Results of radiopharmaceuticaluptake per gram in each tissue/organ were calculated as thepercentage of the total amount of radioactivity administratedby using the following equation.21
% administerd dose=g of tissue ¼ Counts in sample=wt of sampleTotal counts administered
� 100
(1)
Pharmacokinetic parameter, site specific delivery efficiency(SSDE%) was calculated by the following formula:
For spectrofluorometer studies, post samples (6-Cf-CDDP-Imi-Soland 6-Cf-CDDP-Imi-Pts gel) application, 2ml blood sample wascollected by heart puncture from each mouse at 0.5, 1, 2, 4, 6, 8,and 24h, respectively. After collecting the blood sample from eachmouse, the mice were sacrificed and different organs such aslung, liver, spleen, kidney, and skin specimens were collected.All harvested samples were stored at �20 °C and analyzedwithin 1month. Specimens were excised, rinsed, whipped, andhomogenized with a mechanical blender (IKA, Wilmington, NC,USA). The homogenates were then centrifuged at 12,000 rpmfor 15min at 5 ºC and supernatant was vortexed for 2min. Theblood was collected and centrifuged to collect the serum. Thefluorescence content in the serum and isolated organs was thenassayed by spectrofluorometer (Jasco, FP-8300, USA).
Gamma scintigraphic imaging
Swiss albino mice weighing 25–30g were selected for the study.The radiolabeled solution of 99m-Tc-CDDP-Imi-Sol (activity counted:0.3mCi) was administered intraperitoneally at a dose of 200μl/mouse (containing 200μg of cisplatin and 1mg of imiquimod).Similarly radiolabeled formulation of 99m-Tc-CDDP-Imi-Pts gel(activity counted: 0.3mCi) was administered topically on a 2 cm2-shaved area at a dose of 200μl/mouse (containing 200μg of
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Figure 1. Permeation profile of cisplatin. Permeation of cisplatin for CDDP-Imi-Soland CDDP-Imi-Pts gel formulations using mice skin (Mean ± SD, n= 3).
Figure 2. Permeation profile of imiquimod. Permeation of imiquimod for CDDPImi-Sol and CDDP-Imi-Pts gel formulations using mice skin (Mean ± SD, n= 3).
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cisplatin and 1mg of imiquimod). Imaging was performed bysingle photon emission computerized tomography camera(LC 75-005, Diacam, Siemens AG, Erlanger, Germany) at 1-hpost-administration of the radiolabeled samples.
Statistical analysis
All the data are reported as mean± SD and the differencebetween the two groups was tested using Student’s t-test.
Results
Preparation of dual drug formulation for co-delivery ofcisplatin and imiquimod
Cisplatin and imiquimod combination is safe to formulate in aprotransfersome gel formulation that has biphasic compartment(hydrophilic and lipophilic) to reduce its interaction with eachother inwhich hydrophilic drug (cisplatin) has been entrapped intohydrophilic core compartment and lipophilic drug (imiquimod)has been incorporated into lipid bilayer compartment. Carbopol,a potential drug depot for use in controlled-release drug deliverysystems, has enough consistency to be used in topical drugdelivery system.
Evaluation for physico–chemical properties
Viscosity, pH, percentage of drug content, and percentage ofentrapment efficiency of CDDP-Imi-Pts gel formulation are givenin Table 1. The obtained results show a high viscosity value ofCDDP-Imi-Pts gel formulation, which is adequate for the topicalapplication. The pH of the preparation was 6.8 ± 0.17, whichwas equivalent to the dermal pH and physiologically acceptablepH for topical preparations. As expected, the percentage ofEntrapment efficiency (EE) of the cisplatin was found to be lessbecause of reduced availability of aqueous core compartmentfor accommodation, whereas imiquimod entrapment was high,which may be due to a greater availability of lipid to encapsulatethe drug.
Permeation studies
Figures 1 and 2 show the permeation profile of cisplatin andimiquimod from CDDP-Imi-Sol and CDDP-Imi-Pts gel formulationsthrough mice skin model ex-vivo. The data indicate that CDDP-Imi-Pts gel formulation delivers the drug across the skin in constant
Table 1. Physicochemical properties of cisplatin andimiquimod-loaded protransfersome gel formulation
Physicochemical properties
Cisplatin and imiquimod-loaded protransfersome
gel formulation
pH 6.25 ± 0.04Viscosity (cp) 176 ± 4.2TDC for cisplatin (%) 96.89 ± 3.22TDC for imiquimod (%) 98.22 ± 2.32Entrapment of cisplatin (%) 36.22 ± 6.41Entrapment of imiquimod (%) 63.11 ± 3.73
manner. Further, the permeation of cisplatin and imiquimod fromCDDP-Imi-Pts gel formulation was enhanced by approx. 1.5-foldcompared with drug solution (CDDP-Imi-Sol). There was astatistically significant difference (p< 0.05) between CDDP-Imi-Pts gel formulation and CDDP-Imi-Sol for the permeationprofile of cisplatin and imiquimod. Table 2 summarizes the datafor steady-state flux (J), Lag Time (Tlag), and permeabilitycoefficient (Kp).
Radiolabeled and fluorescence-labeled drug formulation
Drug-loaded formulations were successfully radiolabeled andfluorescence labeled using 99m-Tc and 6-CF, respectively.99m-Tc-labeled formulations were optimized for maximumlabeling based on the selection of optimum conditions, that is,pH and concentration of the stannous chloride and incubationtime. The activity counted before and after incubation of99m-Tc-CDDP-Imi-Sol was found to be 1.49 ± 0.011mCi/ml and1.20 ± 0.031mCi/ml, respectively. Likewise activity countedbefore and after incubation of 99m-Tc-CDDP-Imi-Pts gel wasfound to be 1.50 ± 0.021.5mCi/ml and 1.34 ± 0.031.5mCi/ml.The optimized radiolabeled formulations were assessed forin vitro stability in normal saline solution. The percentages of
Table 2. Results of drug permeation parameters for CDDP-Imi-Sol and CDDP-Imi-Pts formulations through mice skin. Results arerepresented as Mean± SD (n=3)
Formulation
Steady-state flux (J) (μg/cm2/h) Permeability coefficient (KP × 10�3) (cm/h) Lag time (Tlag) (h)
cisplatin and imiquimod contents were determined before andafter labeling of the formulations, and subsequently, percentageof radiolabeling efficiency was also determined for cisplatin andimiquimod-loaded formulations. The percentages of cisplatinand imiquimod contents after radiolabeling were found to be96.12 ± 2.28 and 97.96 ± 2.89, respectively, which was almostsimilar with that of before labeling. The percentage ofradiolabeling efficiency of the 99m-Tc-CDDP-Imi-Pts gel and99m-Tc-CDDP-Imi-Sol was found to be 98.64 and 97.98,respectively. Likewise, the percentage of fluorescence labelingof the 6-Cf-CDDP-Imi-Sol and 6-Cf-CDDP-Imi-Pts after purificationfrom the non-labeled markers by dialysis was found to be 98.96and 98.34, respectively. The percentages of cisplatin and
Figure 3. Intraperitoneal administration of99m-
Tc-CDDP-Imi-Sol. Tissue distribution oSD, n= 3) at different time points.
imiquimod contents after fluorescence labeling were found tobe 96.84 ± 1.88 and 98.12 ± 1.92, respectively.
Biodistribution and pharmacokinetic studies
Biodistribution studies of formulations following i.p. (99m-Tc-CDDP-Imi-Sol and 6-Cf-CDDP-Imi-Sol) and topical administration(99m-Tc-CDDP-Imi-Pts gel and 6-Cf-CDDP-Imi-Pts gel) on swissalbino mice were performed, and radioactivity and fluorescenceintensity were measured at different time intervals (i.e., 0.5, 1, 2,4, 6, 8, and 24 h) in various tissues (Figures 3–6). The skin/bloodratios of formulations are recorded in Table 3. The higher valueswere obtained for topical formulations, which reveal the onsite
f99m-
Tc after intraperitoneal administration of99m-
Tc-CDDP-Imi-Sol in mice (Mean ±
-Tc after topical administration of
99m-Tc-CDDP-Imi-Pts gel in mice (Mean ± SD, n= 3)
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Figure 5. Intraperitoneal administration of 6-Cf-CDDP-Imi-Sol. Tissue distribution of 6-Cf after intraperitoneal administration of 6-Cf-CDDP-Imi-Sol in mice (Mean ± SD,n= 3) at different time points.
Figure 6. Topical administration of 6-Cf-CDDP-Imi-Pts gel. Tissue distribution of 6-Cf after topical administration of 6-Cf-CDDP-Imi-Pts gel in mice (Mean ± SD, n= 3) adifferent time points.
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delivery of drugs, thereby potentially preventing the drugs fromreaching the non-target sites. Further, SSDE percentage of valuesof 14417.56 and 137396.56 of radiolabeled and fluorescence-labeled formulations, respectively, is showing higher concentrationof drug at the site administration upon topical application of themarker-labeled formulations.
The pharmacokinetics behavior of 99m-Tc-CDDP-Imi-Sol and 6-Cf-CDDP-Imi-Sol (i.p.) and 99m-Tc-CDDP-Imi-Pts gel and 6-Cf-CDDP-Imi-Pts gel (topical) at a dose of 200μl/mouse (containing 200μg ofcisplatin and 1mg of imiquimod) was assessed after a singletreatment in healthy mice. Table 4 shows the pharmacokineticparameters of radiolabeled and fluorescence-labeled formulations.
The pharmacokinetic parameters (PK) of labeled formulationswerecalculated using one-compartment open model, extravascularadministration methods of residuals. A notably higher maximumactivity (Cmax) in the skin in less time (tmax) with high intensity (AUC)and less activity in blood was found upon topical administration oflabeled formulation in comparison with i.p. administration.
Gamma scintigraphic images
The in vivo SSDE of the radiolabeled formulations for the skinwas evaluated by non-invasive imaging technique using gammacamera that can be effectively used to visualize thebiodistribution of carriers in vivo. Figure 7 shows the scintigramsof the mice at 60-min post-administration of the radiolabeled
samples. As shown in Figure 7(A), 99m-Tc-CDDP-Imi-Sol i.p.treated mice clearly indicate the extensive accumulation of theformulation in the liver and kidney, whereas only a small fractionof the 99m-Tc-CDDP-Imi-Pts gel (topical) was observed in the liver(Figure 7(B)). Figure 7(B) scintigrams demonstrate thatradioactivity at the application point on the mice dorsal surfaceis more intense than the other organs.
Discussion
Cisplatin in combination with imiquimod is safe to formulate inprotransfersome topical drug delivery system to reduce itsinteraction with each other because it has biphasic compartment(hydrophilic as well as lipophilic), where both the drugsseparately accommodate, that is, imiquimod mixed with lipidphase (lecithin) and cisplatin incorporated in aqueous phase(PBS, pH 7.4) during preparation. Moreover, edge activatorcontaining vesicles are flexible in nature. These vesicles are ableto penetrate the skin in a spontaneous manner throughout theintercellular domain. Further at optimum concentration, pluronicf-68 acts as a stabilizer to the vesicular system. In addition,carbopol consists of cross-linked polymer in which the vesicleis entrapped in the gel intramatrix space and releases the drugover a long period. So it is better to choose lipid vesicular topicaldrug delivery system in gel form for cisplatin and imiquimodcombined therapy in cutaneous malignancies.
Cisplatin and imiquimod-loaded protransfersome gel hasfavorable pH and viscosity for dermatological use in terms ofhigher retention and less irritation to the skin. This kind offormulation has a high patient acceptability.
The entrapment efficiency of the CDDP-Imi-Pts gel for cisplatinwas found to be less which may be due to the decreased ratio ofaqueous volume within the vesicles compared with the lipoidalvolume as the drug (cisplatin) did not associate with the lipidbilayers because of it being hydrophilic in nature. However,higher entrapment of imiquimod could be attributed to itshigher phospholipids content. Because imiquimod is lipophilicin nature, the availability of more phospholipids for lipophilicdrug interaction allowed higher amount of drug to besuccessfully entrapped.22 As expected, the permeation of thedrug increased with increase in lipophilicity and entrapmentefficiency of the drug in the CDDP-Imi-Pts gel. Further, thepermeation of the drugs from CDDP-Imi-Pts gel was found tobe more than CDDP-Imi-Sol, which may be due to two reasons.The first is that protransfersome is comprised of elastic vesicles,which are able to penetrate through the skin under the influenceof a transcutaneous hydration force (osmotic gradient). On theother hand, the enhanced permeation from the formulationcontaining isopropanol is possibly because of the branchedchain structure of isopropanol that acts as a co-surfactant andmay have loosened the bilayer packing resulting in better skinpenetration. CDDP-Imi-Pts gel formulation showed less steady-state flux and permeability coefficient but more skin permeationin comparison with drug solution (CDDP-Imi-Sol) because inCDDP-Imi-Pts gel, lipid vesicles may entrap into the intra matrixsystem of the carbopol gel and responsible for the drugaccumulation in deep skin strata. It means that carbopol gelwas found to enhance the skin retention of drugs. Further,they provide higher and sustained skin concentrations ofdrugs.
Biodistribution studies revealed that drug concentration inskin following topical administration of 99m-Tc-CDDP-Imi-Pts gelwas found to be significantly higher (p< 0.05) even at 24 h incomparison with i.p. administration of both 99m-Tc-CDDP-Imi-Sol and 6-Cf-CDDP-Imi-Sol but almost similar to the topicaladministration of 6-Cf-CDDP-Imi-Pts gel, which is an indicativeof site specific delivery of the drug. Thus, CDDP-Imi-Pts geltopical formulations have shown reduced systemic toxicitywhich may be associated with parenteral administration ofanticancer drugs. Results of pharmacokinetics studies indicatethat pharmacokinetics properties are clearly affected by theroute of administration, composition of the delivery system,and probes used for the labeling of the formulation.
The fact of higher activity in the skin upon topical applicationsuggests that carbopol gel has higher retention at the site ofapplication. Its cross-linked structure holds the molecules for along period in intramatrix spaces. Further, longer half-life (t½)was measured during 6-Cf-CDDP-Imi-Pts gel (topical) treatmentin skin in comparison with 99m-Tc-CDDP-Imi-Pts gel because the6-Cf dye is a self-quenching, low molecular weight (376.32),hydrophilic, fluorescence dye. Although 99m-Tc is a metastableradioactive metal. They both have different properties and thatmay be the reason to show different pharmacokinetic parametersin the present investigation.
This may depend on the half-life of technetium-99m (6h). Apartfrom that, longer t½ of drugs for 6-Cf-CDDP-Imi-Pts gel (topical)could be attributed to the reduced uptake of formulation by thereticuloendothelial system.23
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Table 4. Pharmacokinetics parameter of various formulations at different time interval in normal Swiss albino mice
Results are represented as mean± SD (n= 3).CDDP, cisplatin; i.p., intraperitoneal; top., topical.
Figure 7. The images of the mouse injected intraperitoneally with99m-
Tc-CDDP-Imi-Sol (A) and applied topically with99m-
Tc-CDDP-Imi-Pts gel (B) at 60min.99m-
Tc-CDDPImi-Sol (i.p.)-treated mouse clearly indicate the extensive accumulation of the formulation in the excretory organs (liver and kidney), whereas only a small fraction of the99m-
Tc-CDDP-Imi-Pts gel (topical) is observed in the liver.
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The scintigrams clearly demonstrate that the major radioactivitywas seen at the site of application (i.e., skin) 60-min post-topicalapplication of the 99m-Tc-CDDP-Imi-Pts gel as compared with99m-Tc-CDDP-Imi-Sol with respect to other organs, which is justifiedwith biodistribution studies.
Conclusion
A dual drug delivery topical formulation CDDP-Imi-Pts gelcontaining cisplatin and imiquimod could be preparedconveniently for co-delivery of both drugs at the site ofapplication. As per the aforementioned studies, the cisplatinand imiquimod-loaded protransfersome topical gel has retainedsignificant activity on the skin and maintained at a high level.This is remarkably higher than that of the i.p. administration ofboth in solution form.
Studies on topical delivery of CDDP-Imi-Pts gel formulationhave demonstrated that encapsulation in lipid vesicles andintramatrix spaces of gel network system can enhance localizeddrug action, reduced systemic delivery, decreased systemic sideeffects, and toxicity through formulation retention in cutaneousepithelial cells. Additional studies are needed in order to fullycharacterize the nature of tumor uptake and biodistribution as wellas assess the pharmacokinetic studies in diseased or pathologicconditions. Current findings suggest that CDDP-Imi-Pts gelformulation could be a promising modality for solid cutaneousepithelial tumors.
The authors would like express their gratitude to the ViceChancellor, Rajiv Gandhi Technical University, Bhopal, India forproviding the necessary facilities for the research. This work wassupported by the grants from the WOS-A, Department of Scienceand Technology, NewDelhi, India (DST No: SR/WOS-A/LS-36/2010).
Conflict of Interest
The authors did not report any conflict of interest.
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