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Polymer surface-coated nanoparticles for brain targeting

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Double-Coated Poly (Butylcynanoacrylate) Nanoparticulate …

Double-Coated Poly (Butylcynanoacrylate) Nanoparticulate
Delivery Systems for Brain Targeting of Dalargin
Via Oral Administration

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  • 1. Double-Coated Poly (Butylcynanoacrylate) Nanoparticulate Delivery Systems for Brain Targeting of Dalargin Via Oral Administration DEBANJAN DAS, SENSHANG LIN College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, New York, 11439 Received 5 October 2004; revised 23 February 2005; accepted 23 February 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20357 ABSTRACT: The aim of this study is to evaluate oral administration of poly (butylcy- anoacrylate) nanoparticulate delivery systems (PBCA-NDSs), double-coated with Tween 80 and poly (ethylene) glycol (PEG) 20000 for brain delivery of hexapeptide dalargin, an anti-nociceptive peptide that does not cross blood–brain barrier (BBB) by itself. Studies have proven the brain uptake of Tween 80 overcoated nanoparticles after intravenous administration, but studies for brain delivery of nanoparticles after oral administration had been limited due to reduced bioavailability of nanoparticles and extensive degradation of the peptide and/or nanoparticles by gastrointestinal enzymes. To address this problem, dalargin-loaded PBCA-NDS were successively double-coated with Tween 80 and PEG 20000 in varied concentrations of up to 2% each. Measurement of in vivo central anti-nociceptive effect of dalargin along with a dose response curve was obtained by the tail flick test following the oral administration of PBCA-NDSs to mice. Results from the tail flick test indicated that significant dalargin-induced analgesia was observed from PBCA-NDSs with double-coating of Tween and PEG in comparison with single-coating of either Tween or PEG. Hence, it could be concluded that surface coated PBCA-NDS can be used successfully for brain targeting of dalargin or other peptides administered orally. However, further studies are required to elucidate the exact transport mechanism of PBCA-NDSs from gastrointestinal tract to brain. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1343–1353, 2005 Keywords: brain targeting; blood–brain barrier; oral absorption; nanoparticles; peptide delivery; surfactants; dalargin; butylcyanoacrylate; Tween 80; PEG INTRODUCTION epithelia-like tight junctions lining the brain capillary endothelium or the so called blood– Number of individuals who suffer from chronic brain barrier (BBB), more than 98% of all new diseases of the brain is more than the number of potential brain drugs do not cross the BBB.1,2 people stricken with cancer and heart disease In the areas of brain delivery of drugs, there have combined. This large population suffering from been a number of approaches to overcome the chronic brain disorders such as Alzeimer’s, BBB, such as the osmotic opening of tight Depression/Mania, Schizophrenia, Parkinson’s, junctions,3 usage of prodrugs, and carrier systems and HIV infection to name a few, poses the need like targeted antibodies,4 liposomes,5–7 and nano- and opportunity for the growth of brain-targeted particles. For almost a decade, surfactant coated neuropharmaceuticals. Due to the presence of nanoparticles have been reported successfully to transport drugs across the BBB.8–12 Nanoparticle- Correspondence to: Senshang Lin (Telephone (718)-990- 5344; Fax: (718)-990-6316; E-mail: linse@stjohns.edu) mediated drug transport depends on the coating Journal of Pharmaceutical Sciences, Vol. 94, 1343–1353 (2005) of the particles with polysorbates, especially poly- ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association sorbate 80 (Tween 80). Overcoating with these JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005 1343
  • 2. 1344 DAS AND LIN materials leads to the adsorption of apolipopro- in USA.16,31 For the convention of terminology, tein E from blood plasma onto the nanoparticle such nanoparticulate drug delivery systems made surface. The particles then seem to mimic low- with PBCA were termed as PBCA-NDSs. PBCA- density lipoprotein (LDL) particles and interact NDSs were loaded with drug and surface coated with the LDL receptor leading to their uptake by with polyoxyethylene sorbitan monooleate (Tween the endothelial cells lining the BBB.13,14 Then, 80) and poly (ethylene) glycol 20000 (PEG 20000) the drug bound to the nanoparticles may be in varying concentrations of up to 2% each. The released in these cells and diffuse into the interior necessity of Tween 80 overcoat to affect brain or the nanoparticles may be transcytosed. In ad- targeting of nanoparticles has been reported. In dition, it has been suspected that processes such addition to the coating of Tween 80, the second as tight junction modulation or P-glycoprotein coating of PEG 20000 was added. The rationale active efflux system also may occur resulting in of the second coat of PEG (i.e., PEGylation) was brain uptake of nanoparticles. Up to date, many employed for twin reasons. Firstly, PEG was different surfactants15 have been evaluated. Only expected to protect the peptide-loaded nanoparti- Tween 80 overcoat has been able to produce the cles in the hostile gastrointestinal milieu, which most brain targeting effect via intravenous comprises of enzymes and varying levels of pH.19,20 administration16 and the specific role of Tween Secondly, once nanoparticles reach the circula- 80 in brain targeting has also been conclusively tion, PEG was expected to increase the circulation proved.17 However, studies on administration of half-life of the nanoparticles by the ‘‘dysopsonic’’ such nanoparticles orally have been restricted action of the long PEG chains thereby protecting due to the degradation of the drug and/or the it from the rapid clearance by the reticulo- polymer nanoparticles in the gastrointestinal endothelial system and mononuclear macrophage media as well as due to the limited uptake of system.21–24 This investigation was hence, aimed nanoparticles across the gastrointestinal mem- to determine the feasibility of designing PBCA brane. So far, only one study has been reported nanoparticles double-coated with Tween 80 and/or where nanoparticles is administered orally and PEG 20000 for targeted delivery of peptide to brain observed for brain delivery.18 The drug chosen after oral administration. is Leu-enkephalin analog hexapeptide dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg, MW 725.9) which MATERIALS AND METHODS normally does not cross BBB by itself even after intravenous administration.8–11 The anti-noci- Materials ceptive effect produced in mice brain after oral administration of this peptide-loaded nanoparti- The monomer solution containing n-2-butylcya- cles has not been pronounced but rather pro- noacrylate (density 0.9580 at 208C) used for longed.17 Moreover, there was no information on polymerization and fabrication of PBCA-NDSs the dose of dalargin used and the formulation was purchased from Glustitch Inc. (Delta, British development especially designed for delivery of Columbia, Canada). Dalargin (MW 725.9) was nanoparticles through the oral route as well as obtained from CSPS Pharmaceuticals Inc. (San the characterization of nanoparticle formulations Diego, CA). Dextran 70 (MW 68800), naltrexone by measurement of zeta potentials, release profile, HCl, sodium chloride, pepsin, monobasic potas- and stability in simulated gastric and intestinal sium phosphate, pancreatin, Mammalian Ring- fluids. er’s solution (MRS) consisting of sodium chloride The objective of this study was hence aimed 0.96%, potassium chloride 0.04%, calcium chloride at brain targeting of the model peptide drug, 0.03%, sodium bicarbonate 0.02%, and water dalargin, via oral route. For such an objective, a 98.95%; and phosphate buffer solution (PBS) polymeric nanoparticulate drug delivery system consisting of bisodium phosphate/monobasic composed of poly (butylcyanoacrylate) (PBCA) was potassium phosphate/sodium chloride at ratio of fabricated. PBCA nanoparticles are expected to 7.6:1.45:4.8 w/w/w, were obtained from Sigma be biodegraded rapidly in the body without caus- Chemical Co. (St. Louis, MO). PEG 20000, What- ing any significant toxicity. Therefore, long-chain man glass microfiber filters (1.2 and 0.7 m) and alkylcyanoacrylates, such as n-butylcyanoacry- Whatman inorganic membrane Anotop filters late, are commercially available as Indermil1 (0.02 m) were purchased from VWR International and Liquiband1 in Europe, Canada, and USA, (West Chester, PA). Mice (out-bred, albino, female while octylcyanoacrylate markets as Dermabond1 Swiss Websters, 20–25g) were obtained from JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
  • 3. BRAIN TARGETING OF DALARGIN 1345 Taconic Farms (Germantown, NY). Nanopure1 Table 1. Concentrations of Tween 80 and PEG 20000 water (Ultrapure Water System, Barnstead, Used for Double Coating of Dalargin-Loaded Dubuque, IA) was used for the preparation of nan- PBCA-NDSs oparticles. All other reagents were of analytical Formulation Code Tween 80 (%)a PEG 20000 (%)a grade. T0P0 0.0 0.0 T2P2 2.0 2.0 Fabrication, Drug Loading, and T1.5P0.5 1.5 0.5 Double-Coating of PBCA-NDSs T1P1 1.0 1.0 T0.5P1.5 0.5 1.5 Typically, an anionic polymerization method was T0P2 0.0 2.0 followed8–12,14,15,18 using 0.01N HCl solution in T2P2 2.0 2.0 Nanopure1 water. Dextran 70 (1.5% w/w) was a added to it under constant magnetic stirring. Relative to the total suspension volume. Once dextran 70 was completely solubilized in the HCl solution, butylcyanoacrylate monomer solution (1% v/v) was added dropwise. After 4 h of The dalargin-loaded PBCA-NDSs were coated polymerization, the milky nanoparticle solution successively with varying concentrations of up was neutralized with sodium hydroxide (0.1N) to 2% of Tween 80 and PEG 20000 relative to the and the solution was further stirred for 12 h to total suspension volume of nanoparticles (Table 1). ensure complete neutralization. The nanoparticle Depending on the amount of coating of Tween and suspension obtained was subjected to a series of PEG used different formulations such as T1P1 filtration steps using 5, 1.2, and 0.7 m filters by (with 1% of Tween and PEG each) or T2P2 (with means of a vacuum filtration assembly. The 2% of Tween and PEG each) were assigned. For filtered solution was ultracentrifuged for three each formulation, required quantities of Tween cycles, 1 h each at 75600g (Beckman Avanti J-25, and/or PEG were added stepwise in the above Fulerton, CA) with Rotor (Beckman Model Num- solution under continuous magnetic stirring at ber JA 25.50). Finally, the pelleted nanoparticles 9000 rpm for 45 min. Thereafter, the solution was were lyophilized overnight and stored at 48C for centrifuged at 75600g for 20 min, the supernatant drug loading and subsequent surface treatments. containing un-adsorbed drug, as well as excess Drug loading on PBCA-NDSs was done by Tween 80 and /or PEG 20000 was discarded. Then, adsorption method8–10 and was carried out in the double-coated dalargin-loaded PBCA-NDSs 15 mL of MRS, which is better representative of were collected, lyophilized, and stored at 48C for cerebrospinal fluid. The porous nature of PBCA- further use. NDS25 enabled loading of dalargin by continuous stirring of drug with PBCA-NDS in aqueous Characterization of PBCA-NDSs media. Fifty micrograms of lyophilized PBCA- NDS were re-suspended by ultrasonicating at Sample (1 mg) of dried powder obtained from the 4.2 Khz/s for 5 min, which contained dalargin at above step was suspended in 5 mL of Nanopure a concentration of 133 mg/mL. The peptide was water by ultrasonication at 4.2 KHz/s for 5 min. allowed to absorb into the nanoparticle surface for The homogenous suspension obtained was ana- 3 h with continuous magnetic stirring at 9000 rpm. lyzed for particle size, size distribution, and zeta The amount of peptide adsorbed on nanoparticles potential by dynamic light scattering (Nicomp was determined by filtering the suspension 380 DLS, submicron particle-sizer, Santa Bar- through a 20 nm Anotop filter and the amount of bara, CA). A run time for 30 min each was allowed free, un-adsorbed peptide in the filtrate was for each observation, which allowed complete measured by UV spectroscopy. The difference of stabilization of surface charge and hence, lead to total added drug and the amount of free or un- accurate measurements. adsorbed drug gave the amount of drug adsorbed/ entrapped with the PBCA-NDS. All samples were In Vitro Release Kinetics analyzed by UV-VIS-IR spectrophotometer (model number 14NT-UV-VIS-IR, AVIV Instruments, For each formulation, 50 mg of dried powder Lakewood, NJ) at a preset wavelength of 220 nm obtained previously was suspended in 15 mL MRS where a sharp peak, characteristic of dalargin was using ultrasonication described as in previous obtained.8,15 steps. The drug loaded and double-coated PBCA- JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 00, XXXXX 2005
  • 4. 1346 DAS AND LIN NDS was placed in 15-mL screw capped tubes the protected drug after the incubation of 3 h in and kept in a water shaker bath (Thermo Forma, SGF and 12 h in SIF from each formulation were Marietta, OH), which was maintained at 378C and determined. In addition, for the Formulations at 130 cycles per min. A sample volume of 2.5 mL T2P2 (containing 2% Tween 80 and PEG 20000 was collected at predetermined time intervals each) and T0P0 (absence of Tween and PEG), the through 20 nm Anotop syringe filters and the drug stability as a function of time was carried out nanoparticle-free filtrate was analyzed for drug in SGF and SIF, where samples were incubated for content by UV spectroscopy described previously. a specific period of time such as 5 min, 10 min, The sampling regimen had the following pattern: 15 min, 30 min, 1 h, 2 h, and finally 3 h in SGF and every 15 min for the 1st h, every 30 min till the 6th 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and h, every 1 h till the 10th h, every 2 h till the 18th h, finally 12 h in SIF, respectively. Remaining drug in every 4 h till the 34th h, and every 8 h till the end PBCA-NDS after such periods of incubation was of 50th h. detected as described previously. Drug Stability in Simulated Gastric and In Vivo Evaluation of Double-Coated Intestinal Fluids Dalargin-Loaded PBCA-NDSs—Tail Flick Test The stability of peptide loaded PBCA-NDSs with Dalargin, which causes a central analgesic effect or without various coating agents were evaluated in brain by binding with m opioid receptors in simulated gastric fluid (SGF) and simulated for pain perception, was expected to be released intestinal fluid (SIF). SGF and SIF were prepared from dalargin-loaded PBCA-NDS once they were according to USP XXVI. Briefly, SGF was pre- taken up in the brain. Hence, occurrence central pared by dissolving 2 g of NaCl and 3.2 g of pepsin analgesic effect would prove the brain targeting of (derived from porcine stomach mucosa with an PBCA-NDS after oral administration. Groups of enzyme activity of 800–2500 units per mg of ten mice for each formulation were selected. All protein) in 7 mL HCl and finally made up 1000 mL mice were kept at ambient temperature and with adjustment of final pH to 1.2. SIF was humidity conditions with a 12-h light and dark prepared by dissolving 6.8 g of monobasic potas- cycle and fasted overnight. Each mouse was fed sium phosphate in 250 mL water. And then, 77 mL with 1 mL of drug-loaded PBCA-NDSs suspension of 0.2N NaOH, 500 mL of water, and 10 g of by oral gavaging. The dose administered corre- pancreatin were added. The pH was adjusted to sponded to 37.5 mg/kg of mouse body weight, 6.8 Æ 0.1 with 0.2N NaOH and/or 0.2N HCl. which was about five-fold of usual intravenous Pancreatin was obtained as ‘‘Pancreatin Porcine dose for dalargin having central analgesic Pancreas USP’’ containing many enzymes such actions.8 Tail was immersed in hot water main- as amylase, trypsin, lipase, ribonuclease, and tained at 55–608C by a hot plate. The response protease. times, in seconds, taken by each mouse to with- Fifty micrograms of each formulation of PBCA- draw its tail by a sharp ‘‘flick’’ were recorded using NDSs was suspended in 15 mL of either SGF or SIF a stopwatch. The response times were then con- and placed in screw-capped tubes. The tubes were verted to percentage maximum possible effect (% kept in a water shaker bath, which was main- MPE) by method reported elsewhere.14,15 In total tained at 378C and at 130 cycles per min. A specific seven controls and nine formulations were eval- time period of incubation of drug-loaded PBCA- uated (Table 2). Formulation T2P2þA indicates NDS in SGF and SIF were allowed, which were 3 h that naltrexone HCl, an opioid antagonist (A) for SGF and 12 h for SIF, respectively. After these with high oral bioavailability, was co-adminis- time periods, suspensions were centrifuged at tered at a dose of 0.1 mg/kg with Formulation 75600g for 20 min to precipitate the PBCA- T2P2. A perception of pain would signify hence NDS and the supernatants were discarded. The the effect of naltrexone in brain, which displaces precipitated drug-loaded PBCA-NDSs were re- dalargin from its pain receptors. This was done to dispersed in MRS. A rigorous cycle of 20 min of prove the presence of dalargin in brain mainly ultrasonication at 4.2 KHz/s and 5 min of vortexing from the drug-loaded PBCA-NDS targeted to the was subjected towards the nanoparticulate sus- brain as well as to re-establish the fact that pension. Such cycles were carried 20 times to increase of pain threshold was caused only by ensure near complete desorption of drug from the centrally acting and not by peripherally acting PBCA-NDS. Hence, the amounts of remaining or mechanisms. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
  • 5. BRAIN TARGETING OF DALARGIN 1347 Table 2. Formulations of PBCA-NDSs Used in the Tail Flick Test Formulation Code Summary C1 Phosphate buffer solution (PBS) C2 PBS þ Tween (2%) C3 PBS þ PEG (2%) C4 PBS þ Tween (2%) þ PEG (2%) C5 PBS þ drug C6 PBS þ drug þ Tween (2%) C7 PBS þ drug þ PEG (2%) T2P2-N PBS þ drug þ Tween (2%) þ PEG (2%) þ no nanoparticles present T0P0 PBS þ drug þ nanoparticles þ Tween (0%) þ PEG (0%) T2P0 PBS þ drug þ nanoparticles þ Tween (2%) þ PEG (0%) T1.5P.5 PBS þ drug þ nanoparticles þ Tween (1.5%) þ PEG (0.5%) T1P1 PBS þ drug þ nanoparticles þ Tween (1%) þ PEG (1%) T.5P1.5 PBS þ drug þ nanoparticles þ Tween (0.5%) þ PEG (1.5%) T0P2 PBS þ drug þ nanoparticles þ Tween (0%) þ PEG (2%) T2P2 PBS þ drug þ nanoparticles þ Tween (2%) þ PEG (2%) T2P2 þ A PBS þ drug þ nanoparticles þ Tween (2%) þ PEG (2%) þ naltrexone HCl (antagonist) Dose Response Curve of Dalargin calculated on the initial weight of monomer solution used. Other investigators had reported In order to reconfirm the brain uptake and release entrapment efficiency in similar systems to be of dalargin from surface coated PBCA-NDSs, around 25%–30% w/w.8,9 In our study, a higher a dose response study was designed with the mean entrapment efficiency of 39.84 Æ 4.00% w/w Formulation T2P2 that showed the maximum was obtained. The occurrence of higher values of anti-nociceptive effect after dosing. Groups of entrapment efficiency could be attributed to ten mice each were taken and each group was smaller size ranges of nanoparticles (around administered with varying doses of dalargin 100 nm) obtained in this investigation than that from 7.5 to 52.5 mg/kg and observed for anti- (230–260 nm) obtained by other investigators. nociceptive effect after 60 min of dose adminis- Smaller size ranges ensured more available sur- tration. The response times were converted to % face area for the adsorption of the drug on the MPE as described above and a dose response nanoparticle surface. Since the entrapment effi- curve of dalargin was constructed. ciency was about 40% w/w and dalargin was added in the concentration of 133 mg/mL to a Statistical Analysis 15 mL nanoparticle solution containing 50 mg of All results were expressed as mean Æ standard nanoparticles, the amount of drug present in the deviation. A one-way ANOVA test using Statmost pelleted nanoparticles were 798 mg per 50 mg of 3.0 (Datamost Corporation, Sandy, UT) was done to nanoparticles. This amount was used to study the assess any statistically significant difference among in vitro release kinetics and the stability studies the means of % MPE of various formulations of in SGF and SIF. PBCA-NDS in the tail flick test. A post-hoc analysis (Duncan’s Test) was performed to determine the Characterization of PBCA-NDSs groups, which show significant difference. In each case, a p-value less than 0.05 was considered as a All double-coated PBCA-NDSs formulations had representation of significant difference. mean particle sizes of about 100 nm with a low polydispersity index around 0.018. The uniform size range and low polydispersity index obtained RESULTS AND DISCUSSIONS could be attributed to the serial filtration steps employed during the preparation and isolation of Fabrication, Drug Loading, and nanoparticles from the reaction media. It can be Double-Coating of PBCA-NDSs worthwhile to note that effect of double coats of PBCA-NDSs were obtained as a free flowing Tween and/or PEG did not have any significant powder and the yield was found to be 23% w/w effects on the particles size of the nanoparticles. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 00, XXXXX 2005
  • 6. 1348 DAS AND LIN The uncoated particles (Formulation T0P0) had colloidal carriers, preventing their aggregation the similar range of particles sizes as that of in solution. However, since the double-coated double-coated particles with 2% of both Tween PBCA-NDS (Formulation T2P2) had the mean and PEG (Formulation T2P2). Since the nano- zeta potential value of À2.44 mV and other particle diameter did not change significantly formulations with some concentrations of PEG after coating, the exact nature of orientation of showed a positive shift, nanoparticle suspensions Tween and/or PEG molecules upon naked PBCA- prepared with such formulations were prone to NDS needs to be further investigated. However, it particle agglomeration in aqueous media. To re- can be assumed that Tween and/or PEG did not solve this problem, formulations except for For- interact with porous, polymeric PBCA nanoparti- mulation T0P0 were ultrasonicated at 4.2 kHz/s cles, which could have brought about a deviation for a minute to ensure the homogenous dispersion from the constant size ranges of all PBCA-NDSs of nanoparticles. formulations. The mean zeta potentials of different formula- In Vitro Release Kinetics tions varied from À18.01 to À2.44 mVs (Figure 1). The uncoated PBCA-NDS (Formulation T0P0) All formulations showed characteristic biphasic had the highest negative zeta potential value of release with an initial burst release followed by a À18.01 mV and the double coated PBCA-NDSs second phase with a much slower rate of drug with 2% Tween and 2% PEG (Formulation T2P2) release (Figure 2). Release of dalargin from had the lowest negative value of À2.44 mVs. PBCA-NDSs was due to gradual desorption of Interestingly, the zeta potentials of PBCA-NDSs adsorbed drug from the surface of the nanoparti- showed a positive shift with increase in the coat- cles. However, release rate of the drug was ing concentrations of PEG. The shift of the shear different for each formulation suggesting that plane further away from the surface of a nanopar- drug had to diffuse through the polymer and ticulate moiety results in a positive shift of the net surfactant coatings employed upon the PBCA- zeta potential has been reported.17,19,26 Hence, NDSs. After the first 3 h, except for T0P0, all based on the observed trend of positive shift, due to other formulations had almost similar release increasing PEG concentrations, it can be sus- rates. This enforces our findings that outward pected that PEG might cause a shift of shear plane further away from the nanoparticle surface caus- 100 ing the positive shift of the net zeta potentials. It is also worthwhile to note that a high negative zeta potential value is optimal for stabilization of 80 Cumulative Amount Released (%) 60 40 T0P0 T2P0 20 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2 0 0 10 20 30 40 50 Time (hours) Figure 2. Release profile of dalargin from different formulations of PBCA-NDSs over a 50-h time span Figure 1. Zeta potentials of different formulations of (n ¼ 3, error bars were shown only at last two data points PBCA-NDSs (n ¼ 3). to maintain clarity). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
  • 7. BRAIN TARGETING OF DALARGIN 1349 release of drug is indeed a function of polymer 100 and/or surfactant coatings. 90 The highest amount of drug release (82.03 Æ 80 6.33%) at 50-h of release study was obtained from 70 Drug Remaining (%) the PBCA-NDSs without any coating (Formula- tion T0P0). With 2% coating of PEG 20000 60 (Formulation T0P2), the release rate was lowest 50 and was reduced to 50.23 Æ 4.26% over the same 40 period of time. A trend of decrease in release rate 30 with the increase in PEG coating concentration 20 was observed. This trend could be attributed to the fact that the outward release of drug could be a 10 function of coating concentrations of PEG and not 0 of Tween. Drug can be imagined to slowly diffuse T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2 out through the polymer coating, and more the PBCA-NDS Formulations PEG coating concentration, lesser the percentage Figure 3. Stability of dalargin in various formula- release. Some investigators21 have reported that tions of PBCA-NDSs after 3 h of incubation in simulated folding of PEG chains occurring above a certain gastric fluid (SGF) (n ¼ 3). molecular weight form a barrier consisting of con- formationally random PEG chains. Furthermore, such a folding results in unfavorable entropy different formulations were evaluated after 3 h changes, which further results in compression of incubation in SGF (Figure 3), it was observed and stability of the coating layer.21 It can be that percentage of drug remaining was 86.77 Æ imagined the existence of a similar sort of a 1.52% for Formulation T2P2 in comparison to ‘‘barrier’’ caused by increasing coating concentra- 65.38 Æ 2.22% for the Formulation T0P0. This tions of PEG and resulted in the reduction of finding suggests that the percentage of drug pro- release rate. The exact nature of such a barrier tected increased with increasing concentrations formed by random PEG coils needs to be further of PEG coating upon the PBCA-NDSs. A time subjected to structural analysis. dependent stability study for 3 h in SGF (Figure 4) Taking into account the mean zeta potentials, for the Formulations T0P0 and T2P2 further Formulation T0P0 that had the highest amount of showed the protective effect of the PEG coating. release of 82.03% at 50-h also had the most negative zeta potential of À18.01 mV. Formulation T0P2 with the lowest release of 50.23% at the end 100 of 50 h had a near zero zeta potential of À3.61 mV. T2P2 The results indicate that higher the zeta potential, 95 T0P0 higher the release rate at the end of 50 h time span. Thus, it can be surmised that zeta potential had an 90 effect on the release profile of different formula- Drug Remaining (%) tions. These findings perhaps indicate that a high 85 negative zeta potential ensured a better release rate for uncoated formulation than other coated 80 formulations of PBCA-NDS, which might aggre- gate over a period of time and slowed down the 75 drug release rate. 70 Drug Stability in Simulated Gastric 65 and Intestinal Fluids In this investigation, drug-loaded PBCA-NDSs 60 1 2 3 were developed for oral administration, it was Time (hours) important to estimate the protective effect of double coats of PEG and Tween on the labile Figure 4. Comparative stability profiles of dalargin nature of the peptide drug and the polymer. When in Formulations T2P2 and T0P0 in SGF (n ¼ 3). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 00, XXXXX 2005
  • 8. 1350 DAS AND LIN 100 tions T0P0, T2P0, and T0P2 were 65.38 Æ 2.22%, 90 72.66 Æ 1.13%, and 85.02 Æ 1.56%, respectively. 80 Similarly, after 12 h of incubation in SIF, the 70 percentage of drug remaining for Formulations Drug Remaining (%) 60 T0P0, T2P0, and T0P2 were 42.57 Æ 1.16%, 65.02 Æ 50 1.45%, and 75.55 Æ 1.195%, respectively. Results 40 suggest that the enzyme repulsion ability was not 30 only contributed by the PEG but also by the Tween. PEG was well known to form a ‘‘brush,’’ which 20 prevents the docking of enzymes or macrophages 10 on hydrophobic surface of a carrier polymer. It can 0 T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2 hence be hypothesized that the long chains of PBCA-NDS Formulations Tween 80 could have also formed such a protective brush and prevented the degradation of the drug. Figure 5. Stability of dalargin in various formulations The increased surface density of long chained of PBCA-NDSs after 12 h of incubation in simulated molecules such as Tween and PEG was able to intestinal fluid (SIF) (n ¼ 3). exert the protective effect upon the drug-loaded PBCA-NDSs from gastrointestinal enzymes. Similarly, after the incubation of all different formulations in SIF for 12 h (Figure 5), the percen- In Vivo Evaluation of Double-Coated tage of drug remaining increased as a function of Dalargin-Loaded PBCA-NDSs—Tail Flick Test increasing concentrations of PEG coating. More- over, a time dependent stability study of the In this test, time points for all observations Formulations T0P0 and T2P2 (Figure 6) for 12 h spanned for a total of 2 h and at 15 min intervals in SIF also indicated the protective action of (Figure 7). A baseline response time was recorded PEG coating. Thus, in both simulated gastric and using phosphate buffered saline, which served intestinal fluids, it was observed that with as the suspending media for all formulations. increase in PEG 20000 coating, the percentage of drug protected increased. In addition, it was also interesting to note that even with the increase T2P2 T2P2 + A of Tween alone, there had been an increase in 10 0 T0P2 T0.5 P1.5 protective action, but not as significant as that of * T1P1 T1.5 P1.5 PEG. For instance, after 3 h of incubation in SGF, T2P0 T0P0 the percentage of drug remaining for Formula- T2P2 - N 80 100 T2P2 T0P0 90 * 60 MPE (%) 80 Drug Remaining (%) 70 40 * 60 20 50 40 0 30 20 40 60 80 100 120 0 2 4 6 8 10 12 Time (minutes) Time (hours) Figure 7. Percentage MPE of different formulations Figure 6. Comparative stability profiles of dalargin of PBCA-NDSs vial oral administration (n ¼ 10). in Formulations T2P2 and T0P0 in SIF (n ¼ 3). *p < 0.05 when compared to T0P0 at 60 min. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
  • 9. BRAIN TARGETING OF DALARGIN 1351 After 60 min of oral administration, it was ob- PBCA-NDS via oral administration with 2% PEG served that Formulation T2P2 showed the max- coating alone is not possible even though PEG imum anti-nociceptive effect scoring a % MPE of coated PBCA-NDS shows superior protective 93.8 Æ 6.58, followed by T2P0 (60.0 Æ 5.27) and action in simulated gastric and intestinal fluids. T1.5P0.5 (32.5 Æ 6.45). Formulation T2P2þA [i.e., Fourthly, the increase of anti-nociceptive effect T2P2 co-administered with central opioid antago- in terms of % MPE increased as a function of nist (A) naltrexone HCl] showed a near baseline % increasing concentrations of Tween 80 overcoats. MPE of 5 Æ 5.45. A baseline value was observed This claim is supported by the maximum % MPE for the Formulation T2P2-N (i.e., physical admix- attained by different formulations at the 60-min ture of drug and excipients without nanoparticles) time point. For instance, a % MPE of 93.8 Æ 6.58 showing a % MPE of just 2.5 Æ 2.27. Hence, it was was achieved by Formulation T2P2, 60 Æ 5.27 by inferred that co-administration with antagonist Formulation T2P0, 32.5 Æ 6.45 by Formulation naltrexone did not produce a significant anti- T1.5P0.5, and 17.5 Æ 10.54 by Formulation T1P1, nociceptive effect, which was also the case with respectively. This clearly shows that with an the formulation devoid of any nanoparticles. The increase in Tween 80 concentrations, % MPE had maximum effect was observed after 60 min of increased proportionately. Thus, brain delivery of drug administration with return to baseline PBCA-NDS was dependent upon the Tween 80 values at the end of 2 h. Typical Straub Tail coating. effect10 characterized by erect tails at time points of high % MPE were also observed. Statistically Dose Response Curve of Dalargin significant differences ( p < 0.05) were observed between the Formulations T2P2 and T0P0, T2P0 In order to re-establish the phenomenon of brain and T0P0 as well as T1.5P0.5 and T0P0. But, no delivery of dalargin-loaded PBCA-NDS via the statistically significant difference was observed oral route, a dose response curve was obtained between Formulations T1P1 and T0P0. using the Formulation T2P2 (Figure 8). Formula- There could be a number of inferences drawn tion T2P2 was chosen to construct this graph from such observations. Firstly, the brain target- due to the maximum effect of central analgesia ing of dalargin-loaded PBCA-NDSs and release of produced by this formulation in terms of max- drug in the brain interior causing dalargin- imum % MPE of 93.8 Æ 6.58 at the 60-min time induced analgesia was proven. This was confirmed point. The doses increased in aliquots of 7.5 mg/kg by the fact that with co-administration of naltrex- (IV dose) to 52.5 mg/kg (seven times of IV dose). one HCl (Formulation T2P2þA, % MPE of 5.0 at The smallest dose of 7.5 mg/kg failed to show any 60-min time point), a central opioid antagonist, event of dalargin-induced analgesia was absent. Antagonist had more affinity towards the opioid 100 receptors, which had displaced dalargin from its binding sites, enabling mice to feel pain and respond positively to heat stimuli in tail flick test. 80 Other investigators had used naloxone (0.1 mg/kg) in similar experiments,15,18 but in this investiga- MPE (%) 60 tion, naltrexone HCl was used which has greater oral bioavailability than naloxone. Secondly, phy- sical admixture of drug and excipients without the 40 presence of PBCA-NDS (Formulation T2P2-N, % MPE of 2.5 at 60-min time point) failed to elicit anti-nociception, proving that brain delivery of 20 dalargin was only possible when the drug was adsorbed within the nanoparticles. Thirdly, over- coats of PEG, even at 2% concentration (Formula- 0 0 10 20 30 40 50 tion T0P2, % MPE of 7.5 at 60-min time point) was Dose (mg/kg) unable to elicit significant anti-nociceptive effect when compared to 2% overcoat of Tween (For- Figure 8. Dose response curve of dalargin following mulation T2P0, % MPE of 60 at 60-min time point). oral administration of various doses of Formulation This pointed at the fact that brain delivery of T2P2 (n ¼ 10). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 00, XXXXX 2005
  • 10. 1352 DAS AND LIN effect, but with gradual increase of dose to 37.5 mg/ with overcoats of Tween 80 and PEG 20000 kg, a % MPE of 93.75 Æ 5.88 was observed at the represent a feasible method to deliver and target end of 60 min. With a further increase of dose to peptides to brain via the oral route. Although 45 or 52.5 mg/kg, a plateau phase was observed further studies using radioactive markers are with no further increase of % MPE. The graph required to elucidate exact mechanisms of nano- followed a typical sigmoidal curve proving the particular uptake through the gastrointestinal relationship between pharmacodynamic response barrier, polymeric nanoparticles continue to show and the amount of drug released in brain tissue. promise in delivery of macromolecules to complex tissues by traversing biological barriers. CONCULSIONS ACKNOWLEDGMENTS In the light of the success of at least three formu- The authors acknowledge Mr. Vishal Saxena, lations namely T2P2, T1.5P0.5, and T1P1 to cause St. John’s University for his assistance in animal significant dalargin-induced analgesia, it can be concluded that double-coated PBCA-NDS can studies. cross the gastrointestinal barrier after oral ad- ministration and still retain its targeting pro- REFERENCES perties to brain. To summarize, the novelty and success of double-coated PBCA-NDS can be hy- 1. Pardridge WM. 2001. Brain drug targeting: The pothesized due to interplay of a number of factors future of brain drug development, 1st ed. United together. They could be (a) fine particle size of Kingdom: Cambridge University Press, pp 3–11. around 100 nm, (b) near zero zeta potentials, and 2. Pardridge WM. 1998. CNS drug design based (c) double coats of Tween 80 and PEG 20000. The on principles of blood–brain barrier transport. J Neurochem 70:1781–1792. fine particle size of PBCA-NDS could have helped 3. Gummerloch MK, Neuwalt EA. 1992. Drug entry in endocytic uptake, transcytosis across M-cells in into the brain and its pharmacologic manipulation. the gastrointestinal tract.27,28 Also, once is circu- In: Bradbury MWB, editor. Physiology and phar- lation, particles could also escape spleenic filtra- macology of the blood–brain barrier. Handbook tion effect if their size is below 250–300 nm.29 of experimental pharmacology, vol 103. Berlin: Near zero (À2.44 mV Æ 1.18) zeta potentials of the Springer, pp 525–542. Formulation T2P2 could have prevented the 4. Pardridge WM, Buciak JL, Friden PM. 1991. selective adsorption of opsonizing plasma proteins Selective transport of an anti-transferrin receptor and thereby increased the circulation half-life.30 antibody through the blood–brain barrier in vivo. The action of double-coats of Tween and PEG are J Pharmacol Exp Ther 259:66–70. suspected to play the following roles. The role of 5. Zhou X, Huang L. 1992. Targeted delivery of DANN by liposomes and polymers. J Control Rel 19:269– PEG 20000 coating had been the enhancement 274. of stability of drug-loaded in PBCA-NDS in 6. Chen D, Lee KH. 1993. Biodistribution of calcitonin gastrointestinal tract and possibly, mucoadhesive encapsulated in liposomes in mice with particular effect19,31 for better absorption and hence better reference to the central nervous system. Biochem gastrointestinal uptake of nanoparticles. Apart Biophys Acta 1158:244–250. from that, an increase of circulation half-life by 7. Huwyler J, Wu D, Pardridge WM. 1996. Brain drug evasion of the macrophageal clearance of PBCA- delivery of small molecules using immunolipo- NDS in the systemic circulation by dysopsonic somes. Proc Natl Acad Sci USA 93:14164–14169. effect and a PEG mediated uptake of nanoparti- 8. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov cles across BBB can also be considered.32 The AA. 1994. Passage of peptides through the blood– Tween 80 coating can be believed to cause an brain barrier with colloidal polymer particles (nanoparticles). Brain Res 674:171–174. enhancement of oral absorption by temporary 9. Schroeder U, Sabel BA. 1995. Nanoparticles, a drug fluidization of mucus and exposing the nanopar- carrier system to pass the blood–brain barrier, ticles to absorptive enterocytes and the M-cells. permit central analgesic effects of i.v. dalargin Most importantly, as discussed earlier, Tween injections. Brain Res 710:121–124. 80 coating had been responsible for the brain 10. Schroeder U, Sommerfiled P, Ulrich S, Sabel BA. delivery of PBCA-NDS by LDL receptor mediated 1998. Nanoparticle technology for delivery of drugs endocytic uptake across the BBB. Hence, we can across the blood–brain barrier. J Pharm Sci 87: conclude that surface engineered PBCA-NDSs 1305–1307. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
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