Double-Coated Poly (Butylcynanoacrylate) Nanoparticulate
Delivery Systems for Brain Targeting of Dalargin
Via Oral Adminis...
materials leads to the adsorption of apolipopro-
tein E from blood plasma onto the nanoparticle
surface. The particles the...
Taconic Farms (Germantown, NY). Nanopure1
water (Ultrapure Water System, Barnstead,
Dubuque, IA) was used for the preparat...
NDS was placed in 15-mL screw capped tubes
and kept in a water shaker bath (Thermo Forma,
Marietta, OH), which was maintai...
Dose Response Curve of Dalargin
In order to reconfirm the brain uptake and release
of dalargin from surface coated PBCA-NDS...
The uncoated particles (Formulation T0P0) had
the similar range of particles sizes as that of
double-coated particles with...
release of drug is indeed a function of polymer
and/or surfactant coatings.
The highest amount of drug release (82.03 Æ
6....
Similarly, after the incubation of all different
formulations in SIF for 12 h (Figure 5), the percen-
tage of drug remaini...
After 60 min of oral administration, it was ob-
served that Formulation T2P2 showed the max-
imum anti-nociceptive effect ...
effect, but with gradual increase of dose to 37.5 mg/
kg, a % MPE of 93.75 Æ 5.88 was observed at the
end of 60 min. With ...
11. Schroeder U, Sabel BA, Schroeder H. 1999. Diffusion
enhancement of drugs by loaded nanoparticles
in vitro. Prog Neuro-...
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Polymer surface-coated nanoparticles for brain targeting

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

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Polymer surface-coated nanoparticles for brain targeting

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