PHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY

Encapsulation of Valproate-Loaded Hydrogel Nanoparticles in Intact Human Erythrocytes: A Novel Nano-cell Composite for Drug Delivery

MEHRDAD HAMIDI,1,2 PEDRAM RAFIEI,1 AMIR AZADI,1,3 SOLIMAN MOHAMMADI-SAMANI1 1Department of Pharmaceutics, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

2Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanzan, Iran

3Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences,Tehran, Iran

Received 9 May 2010; revised 10 October 2010; accepted 10 October 2010 Published online 24 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22395

ABSTRACT: A novel drug delivery system possessing prolonged release behavior is introduced to the field of carrier erythrocytes and nanotechnology-based drug delivery. Encapsulation of valproate-loaded nanogels inside human erythrocytes as a novel nanocell composite was the objective of the study to obtain a model novel drug delivery system with an intravenous sus- tained drug delivery characteristic. “Ionotropic gelation” was used for the fabrication of hydrogel nanoparticles. The nanoparticles obtained were evaluated in vitro (particle size, transmitting electron microscopy, zeta potential, Fourier transform infrared spectroscopy, etc.). “Hypotonic dialysis” was used to obtain nanoparticle-loaded erythrocytes. Finally, in vitro characterization tests were performed on nanoparticle-loaded erythrocytes. Number- and volume-based sizes, loaded amount (mg), loading ratio (%), and loading efficiency (%) of nanoparticles were, respec- tively, 61 ± 2and74± 2 nm, 20.6 ± 1.02 mg, 31.58 ± 1.86%, and 6.86 ± 0.41%. Spherical structure and slightly negative zeta potential of nanoparticles were confirmed. Erythrocytes were loaded by valproate-loaded nanoparticles (entrapment efficiency of 42.07 ± 3.6%). Carrier erythrocytes showed acceptable properties in vitro and demonstrated a prolonged drug release behavior over 3 weeks. This approach opens new horizons beyond the current applications of carrier erythrocytes for future investigations. © 2010 Wiley-Liss, Inc. and the American Phar- macists Association J Pharm Sci 100:1702–1711, 2011 Keywords: ionotropic gelation; chitosan; hydrogel nanoparticles; sodium valproate; carrier erythrocytes; hypotonic dialysis

INTRODUCTION the term “carrier erythrocyte” was first introduced in 1979 to describe the drug-loading capacity of the The first reports on loading the human living cells by erythrocytes.1 There are a series of advantages en- therapeutic agents for delivery purposes were pub- couraging the wide spread application of erythrocytes lished on carrier erythrocytes idea in 1973.1 Although in drug delivery, including, mainly, biocompatibility, erythrocytes are the most abundant and readily avail- complete biodegradability, long life span in circula- able cells of the human body, they have gained the tion, possibility of targeted drug delivery to the retic- highest and the most extensive attention among other uloendothelial system (RES) organs, relatively simple cellular carriers for time-controlled or targeted deliv- and inert intracellular environment, ease of handling, ery of the drugs and other bioactive agents.1–7 In fact, and so on. One of the most important limitations of the car- Abbreviations used: CS, chitosan; TPP, sodium tripolyphos- phate; TEM, transmitting electron microscopy; RES, reticuloen- rier erythrocytes in drug encapsulation studies is the dothelial system; FTIR, Fourier transform infrared; RBC, red blood rapid escaping of the loaded drug from the carrier- cell; PBS, phosphate-buffered saline; OFI, osmotic fragility index. loaded cells, even during the loading procedure, a Correspondence to: Mehrdad Hamidi (Telephone: +98-241-427- 3638; Fax: +98-241-427-3639; E-mail: [email protected]) problem being worse in the case of lipophilic drugs Journal of Pharmaceutical Sciences, Vol. 100, 1702–1711 (2011) capable of ready diffusion out of the cells. This, © 2010 Wiley-Liss, Inc. and the American Pharmacists Association in turn, makes a big challenge against obtaining

1702 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011 ENCAPSULATION OF VALPROATE-LOADED HYDROGEL NANOPARTICLES IN HUMAN ERYTHROCYTES 1703 carrier cells with desirable drug loading as well as erythrocytes were characterized in vitro for their drug release characteristics, that is, high drug en- drug loading, drug release, and survival-indicating capsulation efficiency with long-term release upon characteristics. reentry to circulation. Several strategies have been attempted to overcome this problem with the use MATERIALS AND METHODS of membrane cross-linkers being the only successful one.8–12 This strategy, however, suffers from the in- Materials clusion of foreign chemicals in the procedure, partic- Chitosan (art. no. 212f498-89 were purchased locally. ularly, considering that these agents are potentially Sodium valproate was kindly donated by Rouz Darou harmful to carrier cells life span in circulation via Pharmaceutical Co. (Tehran, Iran). Dialysis tubing making their membranes more rigid compared with (cutoff 12 kDa; Viskase, Darien, IL, USA) was pur- the normal cells. We, therefore, evaluated a novel chased locally. Other chemicals and solvents were strategy of using drug-loaded nanoparticles, instead from chemical laboratory and high-performance liq- of the “bare” drug for encapsulation in erythrocytes uid chromatography (HPLC) grades, as needed, and to solve the aforementioned problem. were prepared locally. In recent years, significant efforts have been de- voted to use the potentials of nanotechnology in drug Preparation of Valproate-loaded Nanopaticles delivery because it offers a suitable means of deliv- Valproate-loaded nanopaticles were prepared by the ering small as well as large therapeutic molecules ionotropic gelation method, which is based on the or even genes for either targeted or time-controlled ionic interaction between the polycationic polymer purposes.13 The nanometer size ranges of these drug and the sodium tripolyphospahte (TPP).15 The carriers offer certain advantages for drug delivery, the method was optimized by the Taguchi factorial design most profound of them being long circulation time, and the optimized setting was used throughout the and the possibility of selective penetration into tis- study for preparation of nanogels. Briefly, the aque- sues through capillary walls, epithelial linings (e.g., ous solution of sodium acetate with a concentration liver), and being taken up by the cells.14 of 5.54% (w/v, 0.67 M) was prepared and the pH of Hydrogel nanoparticles are a family of partic- the solution was adjusted to 5.5 using glacial acetic ulate polymeric nanostructures (recently referred acid. Then, to this buffer solution, adequate amount of to as nanogels1), being the point of convergence CS was added and mixed thoroughly at 2000 rpm for of the beneficial characteristics of nanoparticles, 2 h to obtain the final concentration of 0.3% (w/v). To as described, and hydrogels, mainly hydrophilicity, prepare the counterion solution, TPP (2.5% w/v) and flexibility, versatility, high water absorptivity, and sodium valproate (25 mg/mL) were dissolved in dis- biocompatibility.15 Besides the commonly used syn- tilled water. The dropwise addition of the anion/drug thetic polymers, active research is focused on the solution to CS bufferic solution with a volume ratio of preparation of nanogels using naturally occurring hy- 1:4 over 2 min at ambient temperature under continu- drophilic polymers. Claiming benefit from these kinds ous stirring (2000 rpm) and leaving the mixture to be of polymers, chitosan (CS)-based nanoparticles are stirred for 20 min resulted in our desired nanoparti- the most widely studied one owing to their remark- cles. Nanoparticles were separated from the rare out- able advantages, which are beyond the scope of this of-range particles by light centrifuging at 3000g for article. CS, the deacetylated derivative of chitin, is 10 min. a US Food and Drug Administration-approved natu- ral gel-forming polycationic polymer with remarkable In Vitro Characterization of Valproate-Loaded advantageous properties for biomedical applications, Nanoparticles including, among others, biocompatibility, biodegrad- Loading Parameters of Valproate in Nanoparticles ability, biosafety, and the capability of forming tailor- made nanoparticles for different purposes.16–19 The loaded amount of valproate in nanoparticles was In this study, CS-based nanogels were prepared, easily determined as follows: loaded by the widely used antiepileptic drug sodium To 1 mL of final nanoparticle suspension, 0.015 mL valproate, a model molecule with high cell-escaping of perchloric acid (HClO4, 70%) was added to destruct characteristics during the erythrocyte encapsulation the nanoparticles. The sample was then vortexed for procedure, optimized, and characterized in vitro and, 1 min and subjected to chromatographic analysis to then, the prepared nanocarriers were encapsulated determine the drug concentration. The result is de- in human intact erythrocytes by hypotonic dialy- fined as total drug concentration. On the contrary, sis method. Finally, the prepared nanogel-loaded a further 1-mL aliquot of the nanoparticle suspen- sion was filtered through a 50-nm filter (Millipore, 1 A registered trademark of Superateck Pharma Inc. (Montreal, Bedfored, Massachusetts) and after adding 0.015-mL Canada) HClO4 (70%) to the filtrate and vortex mixing for

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1 min, the sample was also evaluated chromatograph- the biofate of the carriers, the zeta potential of the ically for quantitation of the free valproate, a value optimized valproate-loaded nanoparticles was mea- known as unloaded drug concentration. A 1:10 dilu- sured using a zetameter (model Zetasizer
R 3000-HS, tion with distilled water was considered on samples Malvern Instruments, Malvern. Worcestershire UK), prior to HPLC analysis to bring the drug concentra- based on the photon correlation spectroscopy. The tion within the linear range of concentrations. measurements were carried out at 25◦C, zeta runs Finally, using the following formulae, the loading of 30, and measurement position of 2 mm. The count parameters of valproic acid in hydrogel nanoparticles rates were 2661.0 and 2730.1 kcps for drug-loaded could be determined: and -unloaded samples, respectively.

Total loaded amount Fourier Transform Infrared Spectroscopy = (total drug concentration For chemical characterization of the drug-loaded −unloaded drug concentration) × 60 nanoparticles, Fourier transform infrared (FTIR) spectroscopy was used. Nanoparticles were freeze- where, the number 60 represents the total volume of dried and, then, their IR spectra were obtained as KBr the final nanodispersion. pellets using a FTIR spectrophotometer (Shimadzu, model 8000 series). The swipe range of the spec- −1 Loading ratio trophotometry was 500–4000 cm in all the cases. = [(total drug concentration unloaded drug Preparation of Nanoparticle-loaded Carrier Erythrocytes −concentration)/total drug concentration] × 100 Preparation of Human Erythrocytes Blood samples were withdrawn by venipuncture Loading efficiency from a healthy male volunteer aged 25 years, using = (total loaded amount/total drug amount 21-G needles connected to disposable polypropy- lene hypodermic syringes and transferred to prehep- added during the procedure) × 100 arinized polypropylene test tubes. After centrifug- ing at 1000g for 10 min, the plasma and buffy Particle Size Distribution coat were separated by aspiration and the remain- To determine the particle size distribution of prepared ing packed erythrocytes were washed three times us- drug-loaded nanoparticles, freshly prepared samples ing phosphate-bufferred saline (PBS; KCl, 2.68 mmol/ of nanoparticles were analyzed using a laser-based L; KH2PO4, 1.47 mmo1/L; NaCl, 136.89 mmol/L; particle size analyzer (Shimadzu, Model SALD 2101, Na2HPO4, 8.10 mmo1/L; pH 7.4). Tokyo, Japan). The instrument worked on photon correlation spectrometry platform and the measure- Encapsulation of Valproate-Loaded Nanoparticles ments were carried out in the particle size range of in Intact Erythrocytes 10 nm–1 mm both in number-based and volume-based A simple and available method was set up in our labo- modes. This measurement was considered as a rou- ratory based on the hypotonic dialysis method widely tine quality control test on all nanoparticles intended used for preparation of carrier erythrocytes with min- to be used in the next procedure involving erythro- imum damage during the drug-loading procedure. As cytes (hypotonic dialysis) on each and every day of the first step, 3 mL of nanoparticle suspension was the experiments. transferred to a dialysis sac, (1.5 × 6cmcutoff12kDa), and to this suspension, 1 mL of washed and packed Transmission Electron Microscopy erythrocytes was added. The dialysis bags were prop- Particle morphology was evaluated using transmis- erly folded and sealed tightly at each end using plas- sion electron microscope (Philips, model CM10, Eind- tic clips. The sacs were inverted 10 times to have hoven, Netherlands). This type of electron microscopy their content properly mixed. Each dialysis bag was is used for the determination of properties such as placed in a container and supported firmly by wedg- shape, aggregation, and internal details. In this step, ing the dialysis clips against the bases. Dialysis was samples of drug-loaded nanoparticles were immobi- performed against 100 mL of hypo-osmotic phosphate lized on copper grids, dried at room temperature, and buffer (KH2PO4, 5 mmol/L; K2HPO4, 5 mmol/L, pH were ultimately subjected to the instrument without 7.4) at 4◦C in an ice bath, with very gentle magnetic being stained. stirring speed (less than 10 rpm). The bags remained in this condition for 1.5 h. Particle Zeta Potential In the annealing step, dialysis sacs of previous step Because the surface zeta potential of the drug-loaded were transferred to a container of 100 mL eutonic PBS nanoparticles is one of the major determinants of having sodium valproate concentration of 10 mg/mL

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011 DOI 10.1002/jps ENCAPSULATION OF VALPROATE-LOADED HYDROGEL NANOPARTICLES IN HUMAN ERYTHROCYTES 1705 with adjusted temperature at 37◦C, with very gentle several gentle inversions. Then, the mixture was di- magnetic stirring speed (less than 10 rpm). The time vided into 1.5-mL portions in 1.5 mL of polypropy- of this step was 30 min. lene microtubes. The samples were shaken vertically The resealing procedure was done by transfer- (15 rpm) while incubated at 37◦C using a vertically ring dialysis bags to a container of 100 mL hyper- shaking incubator designed and assembled in-house. tonic phosphate buffer (KCl, 2.68 mmol/L; KH2PO4, At the beginning of the test and at 0.25, 0.5, 1, 2, 4, 1.47 mmo1/L; NaCl, 273.78 mmol/L; Na2HPO4,8.10 10, 20, 48, 72, 120 hr, and 1, 2, and 3 weeks intervals, mmo1/L; pH 7.4) having sodium valproate concentra- one of the aliquots was harvested and after centrifug- tion of 10 mg/mL with adjusted temperature at 37◦C, ing at 1000 g for 2 min, a part of the supernatants with very gentle magnetic stirring speed (less than was separated for valproate assay. In addition, the 10 rpm) for 30 min. absorbance of another portion of the supernatants Finally, the content of dialysis sacs was transferred was determined at 540 nm using a ultraviolet (UV)/ into propylene tubes and the carrier erythrocytes ob- visible spectrophotometer (Cecil, model Series 9000, tained by this manner were washed three times us- Cambridge, UK) to monitor the hemoglobin release. ing 10-mL aliquots of PBS followed by centrifugation These experiments were carried out in triplicate and at 1000g for 10 min to wash out the unentrapped the percent of valproate and hemoglobin release were nanoparticles, free sodium valproate, the released determined in reference to a completely lysed sample hemoglobin, and other cell constituents released dur- (100% release), which was prepared by adding dis- ing the loading process. tilled water instead of Ringer solution to one replicate In some experiments, valproate-loaded erythro- of the above-mentioned samples. cytes were needed for comparison that was prepared as described except for using the equi-concentration Osmotic Fragility free drug solution instead of drug-loaded nanopar- To evaluate the resistance of erythrocytes’ mem- ticles in initial sac filling. Also, sham-enacpsulated branes against the osmotic pressure changes of samples were prepared as described, but without any their surrounding media, 0.1-mL aliquots of the drug or nanoparticles in process. packed samples of each type of erythrocytes, Loading Parameters that is, nanoparticle-loaded, valproate-loaded, sham- encapsulated, and intact (uloaded) erythrocytes were To evaluate the final drug-loading efficiency of the suspended in 1.5 mL of NaCl aqueous solutions hav- prepared erythrocyte carriers, two indices were de- ing the osmolarities of 0–300 mOsm/L (10 concen- fined as loading parameters: trations). After gentle vertically shaking at 37◦Cfor 15 min, the suspensions were centrifuged at 1000 g (i) Loaded amount, the amount of valproate en- for 5 min and the absorbance of the supernatants trapped in 1 mL of the final packed erythrocytes. determined spectrophotometrically at 540 nm. The (ii) Efficiency of entrapment, the percentage ratio of released hemoglobin was expressed as percentage ab- the loaded amount of valproate to the amount sorbance of each sample to a completely lysed sam- expected if the drug was encapsulated with no ple prepared by diluting 0.1 mL of packed cells of limitation and remained inside the cells com- each type with 1.5 mL of distilled water instead of pletely upon final washing. NaCl solutions. For comparative purposes, an osmotic fragility index (OFI) was defined in each case because In Vitro Characterization of Nanoparticle-Loaded the NaCl concentration producing 50% hemoglobin Human Carrier Erythrocytes release.4 A series of tests were carried out to characterize the nanoparticle-loaded erythrocytes in comparison with Turbulence Fragility the unloaded, sham-encapsulated, and the free drug- To exploit the mechanical strength of the erythro- loaded ones. Sham-encapsulated cells were erythro- cytes’ membranes, 0.5 mL samples of erythrocytes of cytes undergoing loading conditions only without the each group were suspended in 10 mL of PBS and were drug or nanoparticles. shaken vigorously using a multiple test tube orbital shaker (IKA, model VIBRAX VXR basic, Staufen, Drug and Hemoglobin Release Germany) at 2000 rpm for 20 h. To determine the time To exploit the release kinetic of valproate as well as course of hemoglobin release, 0.5 mL portions of each hemoglobin (an indicator of cell lysis) from carrier ery- suspension were withdrawn in 0, 0.5, 1, 2, 4, 8, 12, throcytes, 1 mL of packed nanoparticle-loaded cells 16, 18, and 20 h, and after centrifuging at 1000g for was diluted with 6.5 mL Ringer solution containing 5 min, the absorbance of the supernatant was deter- 0.01% sodium azide (NaN3), an antimicrobial preser- mined spectrophotometrically at 540 nm. The percent vative, and the suspension was mixed thoroughly by release of hemoglobin was determined in reference to

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011 1706 HAMIDI ET AL. a completely lysed cell suspension with the same cell statistics approach. Whenever the standard devia- fraction (i.e., 0.5 mL of packed cells added to 10 mL tions were reported, the experiments were carried out of distilled water). in triplicate (n = 3). Hematological Indices The hematological indices of four types of erythro- RESULTS AND DISCUSSION cytes, that is, nanoparticle-loaded, valproate-loaded, Preparation of Valproate-Loaded Nanoparticles sham-encapsulated, and unloaded packed erythro- cytes obtained from the same subject were deter- As the starting point, we tried to encapsulate valproic mined using a coulter counter-based instrument acid in carrier erythrocytes using the gentle dialysis (Hematology, model MS9, Stockholm, Sweden). The method described in Materials and Methods.Nosig- tested parameters consisted of mean corpuscular vol- nificant amount of the drug was detected in the fi- ume, mean corpuscular hemoglobin, and mean cor- nal resulting erythrocytes that was confirmed by the puscular hemoglobin content. presence of the drug quantitatively in final three-step wash solutions, which indicated that the drug leaves Laser-Assisted Particle Size Analysis the erythrocytes during the washing steps. It can be To investigate the effect of loading process on best explained by the lipophilicity and the size of the the particle size distribution of the erythrocytes drug, which favors the passive diffusion out of the population, a laser-based particle size analysis cells. Therefore, to solve this problem, we developed (Shimadzu, model SALD-2101) was used. For this the current novel approach to encapsulate the drug- purpose, 20 :L of nanoparticle-loaded, valproate- loaded nanogels, instead of the free drug, in the ery- loaded, sham-encapsulated, and unloaded packed throcytes. In this way, hydrogel nanoparticle was pre- erythrocytes were analyzed while suspended in saline pared using the ionotropic gelation method described in a dilution according to instrument operation con- earlier in this article, with the method parameters ditions. optimized according to a Taguchi factorial design ap- proach, considering particle size as the main response Drug Assay factor. The detailed methodology and results of this The concentrations of valproate in different samples part are beyond the scope of the current paper and throughout the study was determined by a simple will be published as a separate work. Using this ap- reversed-phase HPLC method developed and vali- proach, particles with diameters less than 100 nm dated in initial phase of the study. In brief, the chro- were obtained (detail features in next section). The optimized setting was evaluated for validity and the matographic system consisted of a C18 column (100 × 4.6 mm; Merck) with a precolumn guard with the results showed that the method is highly robust with same packing, and a binary mixture of phosphate particles having sizes in a narrow range as shown in buffer (0.025 M, pH 6) and acetonitril (60:40) as corresponding section in next part of this paper. The stationary and mobile phases, respectively. A pump- average loading parameters of valproate in nanopar- controller unit (Knauer, Smartline
R , model 1000, ticles are shown in Table 1. These data indicate that Berlin, Germany) and a Rheodyne injection device the drug has significant absolute and yield of load- equipped with a 50 :L loop were used for solvent ing in nanogels. The dose sufficiency of the loaded delivery (flow rate 1 mL/min) and sample injection, nanoparticles can be judged only after preclinical and respectively. The analyte detection was made by a clinical studies because the pharmacokinetic features UV detector (Knauer, model 2500) at wavelength of of the drug upon administration of these nanocarriers 210 nm. The chromatograms were processed using to host organism is predicted to be completely differ- compatible software (Knauer, Eurochrom
R ). ent from the conventional free drug, mainly depend- In the case of the erythrocyte lysate samples in- ing on the biofate of the nanoparticles. To avoid the tended to be assessed for drug content, the chro- misinterpretation of the data, we checked the pres- matographic system was the same except for the bi- ence of any particles in filtrates upon passing through nary mixture of phosphate buffer [0.435% (w/v), pH 50-nm filter and confirmed that no significant amount 5.5]: acetonitril (80:20) and its flow rate (2 mL/min). of particles in the range of our particle size analyzer detection, that is, >0.6 nm was evident in filtrates. Twenty microliter of 70% HCLO4 was added to 1 mL of erythrocytes and after vortex mixing for 1 min, the mixture was centrifuged in 4000g for 15 min and the Table 1. Loading Parameters of Valproate in Nanoparticles supernatant was used for drug analysis. Parameter Mean ± SD Statistics Loaded amount (mg) 20.6 ± 1.02 ± No analytical statistics was used in this study. All Loaded ratio (%) 31.58 1.86 Loading efficiency (%) 6.86 ± 0.41 the data presented are being used as a descriptional

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011 DOI 10.1002/jps ENCAPSULATION OF VALPROATE-LOADED HYDROGEL NANOPARTICLES IN HUMAN ERYTHROCYTES 1707

Table 2. Distribution of Size and Zeta Potential of Hydrogel Nanoparticles

Zeta Potential (mv) Sizea (nm)

Unloaded nanoparticles Drug-Loaded Nanoparticles Volume-based Number-based −9.01 −6.80 74 ± 261± 2

avalproate-loaded nanoparticles.

In Vitro Characterization of Valproate-Loaded the prepared nanoparticles for the long-circulating Nanoparticles intravenous delivery, that is, around zero zeta. The fact that our particles show slightly negative zeta po- Distribution of size and zeta potential of hydrogel tential may be explained by the superiority of the con- nanoparticles is presented in Table 2. The mean sizes tribution of the phosphate groups of TPP in surface of 61 nm for number-based and 74 nm for volume- structures resulted, in turn, from the higher molar lo- based diameters that were obtained are not highly calization of phosphate ions in the experimental con- different from each other, thus indicating a relatively dition. The drug loading has clearly shifted the zeta homogenous “in-range” unimodal particle population. potential of nanoparticles toward positive, which was This size range, although being desirable in a gen- unexpected considering the dissociated, that is, nega- eral sense for intravenous drug delivery (for being ig- tively charged nature of valproate in pH of the loading nored by phagocyting RES), seems to be suitable for condition. Although the extent of difference between the next step, that is, for being used as a carrier for the zeta potentials of these two types of naoparticles drug encapsulation in erythrocytes. Figure 1 demon- is marginal and thus not noteworthy, it needs more strates the transmission electron microscopy (TEM) mechanistic evaluation by further studies. There are image of valproate-loaded nanoparticles. The size ob- a series of reports on nanoparticles obtained via the tained by laser-scattering approach can be clearly same preparation method indicating the zeta poten- confirmed by this finding along with a direct evidence tial of slightly positive.22–25 of the spherical uniform shape of nanoparticles indi- In characterizing the FTIR spectra (Figs. 2 and cating main spherical particles surrounded by some 3), there are primarily three determinant peaks of smaller “sattelite” particles. Various studies have also CS around 3400 cm−1 for -OH groups, 1100 cm−1 20,21 −1 reported spherical shapes but the evidences from for C-O-C, and 1600 cm for -NH2 groups. As ex- the formation of polyhydrons, instead of spheres, is pected, the peak at 3400 becomes wider as TPP be- published.22 comes associated with CS in nanostructures, which The zeta potential distribution diagrams of the can be attributed to the resulting enhanced hydrogen valproate-loaded as well as -unloaded nanoparti- bindings. In addition, in CS–TPP nanoparticles, the cles demonstrated an overall mean zeta potential peak of 1600 cm−1 for -NH2 bending vibrations shifts of slightly negative, that is, −6.80 and −9.01 mv to around 1500 cm−1 and a new sharp peak at 1630 for valproate-loaded and -unloaded nanoparticles, re- cm−1 appears, which can be explained by the CS film spectively. These values, although deserving further modified by phosphate in the form of the association improvements, again emphasize the applicability of between phosphate and ammonium ions. Therefore,

Figure 1. Transmission electron microscope image of valproate-loaded nanoparticles.

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Figure 2. Fourier transform infrared spectrum of sodium valproate. it can be concluded that the phosphate groups of via electrostatic interactions with the polymer TPP are linked with ammonium groups of CS in structure. nanoparticles formation. Compared with the spec- Our FTIR results (Fig. 3) are in agreement with trum of sodium valproate (Fig. 2), in the spectrum studies published by Wu et al.25 and Xu and Du.26 of sodium valproate-loaded nanoparticles, the absorp- tion peak of about 1550 cm −1 (carboxyl group) disap- In Vitro Characterization of Carrier Erythrocytes − pears and a new shoulder peak of 1453 cm 1 (carboxyl The final loading parameters of valproate in the pre- salt) appears. This confirms the presence of the elec- pared nanocell composite are shown in Table 3. Con- trostatic interactions between carboxyl groups of val- sidering the rapid leaking of the free valproate out proate and amino groups of CS. Therefore, valproate of the erythrocytes in our initial phase studies, this molecules are, at least in part, loaded in our nanogels result is promising by showing that the drug remains

Figure 3. Fourier transform infrared spectra of (a) unloaded and (b) sodium valproate-loaded nanoparticles.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011 DOI 10.1002/jps ENCAPSULATION OF VALPROATE-LOADED HYDROGEL NANOPARTICLES IN HUMAN ERYTHROCYTES 1709

Table 3. Loading Parameters of Valproate in Human Carrier Erythrocytes

Parameter Mean SD CV % Loaded amount (mg/mL) 0.435 0.031 7.0 Entrapment efficiency (%) 42.07 3.68.6 inside the cells as loaded in nanoparticles in such a way that more than 40% of the drug amount, which is available for loading during the procedure, is encap- sulated. The dose sufficiency of this amount needs to be further evaluated in vivo. Comparing the results of drug-loading parameters in bare nanoparticles and Figure 5. Osmotic fragility curves of four types of ery- in composite form, and considering that the nanopar- throcytes. ticles are transferred to dialysis bags as prepared, that is, nanodispersions, it is evident that the loading efficiency of the drug in composite obeys that of the barrier” role of erythrocytes needs to be identi- nanoparticles. In other words, the drug retention in fied in further mechanistic studies. erythrocytes seems to be solely via the drug-loaded nanoparticles, as expected. Thus, our composite sys- The OFIs of unloaded, sham-encapsulated, tem has successfully fulfilled its goal of drug loading valproate-loaded, and nanoparticles-loaded ery- in erythrocytes mediated by a secondary reservoir. throcytes, being 185.0, 182.5, 182.5, and 186.3, From the drug and hemoglobin release profiles respectively, indicates that the loading procedure demonstrated in Figure 4, the following conclusions with nanoparticles results in no significant deviations can be drawn: from the natural cell membrane osmotic integrity (Fig. 5). In addition, the loading process alone or with naked drug, also, has no significant destructing effect (i) Our composite loading strategy seems to be com- on cells. pletely successful by retarding the drug release In most of the studies testing this parameter, the os- from rapid drug escaping during carrier prepa- motic fragility curves indicate that dialyzed/resealed ration to a slow release trend up to our final erythrocytes are slightly more resistant to hypotonic sampling point of 3 weeks, which is an accept- hemolysis than the normal cells.27,28 able time for an intravenous delivery system. Turbulence fragility curves of nanoparticles-loaded (ii) The release profile of valproate seems to be con- erythrocytes (Fig. 6) compared with the normal un- sistent to that of hemoglobin, indicating that loaded cells indicate a remarkable increase in cell the drug can be released only subsequent to cell resistance against the vigorous mechanical stresses lysis, an interesting beneficial finding with re- even after 20 h of shaking. The higher mechanical re- spect to the aim of the current study, but with no sistance can be attributed, at least in part, to the con- explanation of the retention of the drug released tribution of flexible hydrogel structures of nanoparti- from nanoparticles inside the erythrocytes until cles in compensating the external forces exerted to the the cell destruction. This “secondary reversible cell membrane. The results for sham-encapsulated cells show that the encapsulation procedure is partly responsible for this resistance-increasing effect. In contrast, the results of other studies have shown that turbulence fragility of the carrier erythrocytes in- creases significantly in comparison with the normal unloaded cells.1,3,4,7 From the data in Table 4, no significant changes are evident in three hematological indices of nanoparticles-loaded erythrocytes, as expected from the gentle nature of the loading method used. How- ever, these differences are higher in the case of sham- encapsulated and drug-loaded cells. Again, some cell-protecting effect of hydrogel structures becomes Figure 4. Release profiles of valproate and hemoglobin evident. In other studies,27,29,30 all these parameters from carrier erythrocytes loaded by valproate-loaded were decreased in carrier erythrocytes compared with nanoparticles. the normal unloaded cells.

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Table 4. Hematological Indices of Four Types of Erythrocytes

Sham-Encapsulated Drug-Loaded Nano-Loaded Hematological Index Packed Erythrocytes Erythrocytes Erythrocytes Erythrocytes MCV (fL) 89.90 (0.28)a 76.50 (2.16) 81.27 (4.79) 89.77 (0.34) MCH (pg) 29.67 (0.96) 21.97 (1.23) 25.47 (2.96) 29.80 (0.29) MCHC (g/dL) 33 (0.99) 28.77 (0.1.28) 31.27 (1.87) 33.27 (0.17)

aMean (SD). MCV, mean corpuscular volume; fL, femtoliter; MCH, mean corpuscular hemoglobin; pg, pictogram; MCHC, mean corpuscular hemoglobin content; g/dl, gram per deciliter.

Table 5. Statistical Parameters of Number-Based Size Distribution of Different Types of Erythrocytes

Statistical Sham-Encapsulated Drug-Loaded Nanoparticle-Loaded Parameters Packed Erythrocytes Erythrocytes Erythrocytes Erythrocytes Mean (:m) (SD) 4.72 (0.086) 4.47 (0.094) 4.37 (0.097) 4.68 (0.085) Median (:m) 4.65 4.40 4.31 4.61 Mode (:m) 4.47 4.47 4.47 4.47

Figure 6. Turbulence fragility curves of four types of erythrocytes.

From the size distribution of the erythrocytes rep- lowing it to be regarded as a promising potential for resented by the data in Table 5: first, the erythro- long-circulating drug delivery. Regardless the phar- cytes showed normal unimodal distribution in all four macokinetic outcome of valproate upon in vivo evalu- groups; second, the sizes correspond the mean equiv- ation of this new carrier, the drug can be regarded as alent diameters of normal erythrocytes; third, en- a model and, thus, this study can serve as an initial capsulation of nanoparticle-loaded valproate in ery- point for a series of studies on this delivery system throcytes has no significant effect on cell sizes; and as a candidate for long-life drug delivery in systemic fourth, the encapsulation procedure alone exerts no circulation. changes in cell sizes. These observations can, again, be attributed to the gentle nature of the loading method. REFERENCES 1. Hamidi M, Tajerzadeh H. 2003. Carrier erythrocytes: An overview. Drug Deliv 10:9–20. CONCLUSION 2. Hamidi M, Zarrin A, Foroozesh M, Mohammadi-Samani The results of this study indicate that valproate can S. 2007. Applications of carrier erythrocytes in delivery of biopharmaceuticals. J Control Rel 118:145–160. be loaded significantly in hydrogel nanoparticles via 3. Hamidi M, Zarei N, Zarrin AH, Mohammadi-Samani S. 2007. geometric, if any, and electrostatic interactions and Preparation and in vitro characterization of carrier erythro- the resulting nanocarriers can be encapsulated in hu- cytes for vaccine delivery. Int J Pharm 338(1–2):70–78. man intact erythrocytes to obtain a nanocell com- 4. Hamidi M, Zarrin AH, Foroozesh M, Zarei N, Mohammadi- posite with acceptable drug-loading parameters. In Samani S. 2007. Preparation and in vitro evaluation of carrier erythrocytes for RES-targeted delivery of interferon-alpha 2b. vitro characteristics, overall, showed that this deliv- Int J Pharm 341(1–2):125–133. ery system having remarkable slow-release profile of 5. Hamidi M, Zarei N, Zarrin A, Mohammadi-Samani S 2007. the drug is highly near-to-normal cell properties al- Preparation and validation of carrier human erythrocytes

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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 5, MAY 2011