Developing the Potential Ophthalmic Applications of Pilocarpine

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Developing the Potential Ophthalmic Applications of Pilocarpine Entrapped Into Polyvinylpyrrolidone−Poly(acrylic acid) Nanogel Dispersions Prepared By γ Radiation Hassan A. Abd El-Rehim,*,† Ahmed E. Swilem,‡ Anke Klingner,§ El-Sayed A. Hegazy,† and Ashraf A. Hamed‡ †

Department of Polymers, National Center for Radiation Research and Technology, Nasr City, Cairo 11371, Egypt Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo 11566, Egypt § Department of Physics, Basic Science, German University in Cairo, Al−Tagmoa Al−Khames, New Cairo 13411, Egypt ‡

ABSTRACT: The aim of this study was to improve the stability and bioavailability of pilocarpine in order to maintain an adequate concentration of the pilocarpine at the site of action for prolonged period of time. Thus, pH-sensitive polyvinylpyrrolidone−poly(acrylic acid) (PVP/PAAc) nanogels prepared by γ radiationinduced polymerization of acrylic acid (AAc) in an aqueous solution of polyvinylpyrrolidone (PVP) as a template polymer were used to encapsulate pilocarpine. Factors affecting size and encapsulation efficiency were optimized to obtain nanogel suitable for entrapping drug efficiently. The PVP/PAAc nanogel particles were characterized by dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM), and their size can be controlled by the feed composition and concentration as well as the irradiation dose. Pilocarpine was loaded into the nanogel particles through electrostatic interactions where the AAc-rich nanogels exhibited the highest loading efficiency. The transmittance, mucoadhesion, and rheological characteristics of the nanogel particles were studied to evaluate their ocular applicability. The in vitro release study conducted in simulated tear fluid showed a relatively long sustained release of pilocarpine from the prepared PVP/PAAc nanogel particles if compared with pilocarpine in solution.

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INTRODUCTION Nanotechnology is an emerging technology with enormous potential in area of biotechnology and medicine applications that hold the promise of providing great benefits for society in the future. Novel nanobiomaterials and nanodevices are fabricated and controlled at the nanoscale size because the matter functions differently from both the individual atomic and the macroscopic scales. Among the different application areas of nanotechnology, hydrogel nanoparticles have gained considerable attention in recent years as one of the most promising soft nanoparticles for drug delivery.1−6 Fast response to environmental factors, large surface area, and stable network structure are the main characteristic properties making nanohydrogels suitable for this purpose. In recent years, significant efforts have been devoted to use the potentials of nanoparticles in ocular drug delivery.7−9 Most ocular diseases are treated with drug solutions administered as eye drops. These solutions are usually highly concentrated and require frequent application due to normal ocular protective mechanisms such as blinking and tear drainage that promote rapid clearance and reduced bioavailability. Nanoparticles have provided an effective way to overcome these drawbacks. The primary motivation for using nanoparticles is their use in liquid © 2013 American Chemical Society

form, just like eye drops; therefore, they do not produce the discomfort caused by viscous or sticky ointments.10 Nanoparticles based drug delivery systems in ophthalmic applications have the ability to prolong drug residence times by trapping the drug in the ocular mucus layer.11 Some papers based on nanohydrogels as a platform for new ocular drug delivery appeared in the literature.12,13 For the ocular therapy of most glaucoma patients, polymeric nanoparticles are being developed to find a suitable carrier for pilocarpine and to increase its efficacy, stability, and bioavailability.14−18 Pilocarpine, as a natural parasympathomimetic alkaloid, is an imidazole derivative with a pKa of 7.2 and exhibiting pharmacological activity.19−21 Pilocarpine has been widely used topically in the eye for the treatment of open-angle and other chronic glaucomas. The major problem with the pilocarpine eye drops is that the drug is delivered immediately after instillation, followed by rapid decline to insignificant levels, usually within minutes. This means that pilocarpine suffers from low ocular bioavailability. Therefore, it is used Received: November 9, 2012 Revised: December 26, 2012 Published: February 15, 2013 688

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was purchased from Sigma-Aldrich (U.S.A.). Other chemicals were of analytical grade. Deionized water was used in all the experiments. Preparation of PVP/PAAc Hydrogel Nanoparticles. PVP/ PAAc hydrogel nanoparticles were prepared via γ radiation-induced polymerization of AAc in an aqueous solution of PVP as a template polymer. Different compositions of PVP/AAc mixtures were dissolved in deionized water at a total concentration of 1.5 w/v % and then subjected to γ-rays generated from a 60Co source with a dose rate of 3.7 kGy/h. The irradiation process was carried out at ambient temperature under an air atmosphere. To calculate the yield of nanoparticles, the colloidal nanogel suspensions were ultracentrifuged (Sorvall Ultra 80, U.S.A.) at 20000 rpm for 30 min at 4 °C. Supernatants as well as aggregated nanogels were collected and freezedried in order to determine the weight of the polymers that formed nanogels. The production yields were calculated from the mass ratio of polymers forming nanogel and the polymer and monomer initially introduced in the preparation procedure. To determine the level of residual acrylic acid or soluble PVP and PAAc in irradiated nanogel products, an aliquot of the supernatant was injected onto high performance liquid chromatography and gel permeation chromatography columns. Loading of Pilocarpine. The prepared PVP/PAAc hydrogel nanoparticles were neutralized by NaHCO3 solution, ultracentrifuged at 40000 rpm and 4 °C for 45 min, and finally freeze-dried to obtain pure and easy dispersible dry nanogels. The freeze-dried nanogel particles were dispersed in deionized water by sonication at a concentration of 0.5% w/v. Pilocarpine hydrochloride powder was then added to the nanogel dispersion at 1/5 weight ratio of drug to nanogel, followed by incubation for 48 h with continuous magnetic stirring at room temperature. The drug-polymer solutions were then ultracentrifuged to separate the drug-loaded nanogel particles. The concentration of free pilocarpine in the supernatant was measured spectrophotometrically at 215 nm after appropriate dilution with deionized water. The loading efficiency of pilocarpine into the nanogel particles was calculated using the following equation:

usually up to 4 times daily, which may lead to compliance failures, as well as an increased risk of side effects.22 Moreover, the aqueous solution of pilocarpine is subjected to chemical degradation. For an optimal stability, the eye drops are formulated at a pH between 4 and 5. However, the drug is almost ionized at these pH values and its ocular absorption is reduced.23,24 It is known that the complex formation between drugs and some macromolecules through van der Waals forces, hydrogen bonding, dipole−dipole interactions, and hydrophobic interactions often leads to stabilization of the active drug components and prolonging residence time of aqueous solution in the eye.25 Poly(acrylic acid) hydrogel is an anionic mucoadhesive polymer widely employed in ophthalmic formulations. It has ionizable carboxylic groups that allow interactions with oppositely charged drugs through the formation of a polyelectrolyte−drug complex.26,27 These properties can modify and control drug delivery and increase the low soluble drugs compatibility.28,29 Additionally, it has been reported that the interaction of poly(acrylic acid) with the basic drugs as lidocaine and azythromycin enhances their chemical stability in aqueous solutions.26,30 However, the high viscosity of poly(acrylic acid) hydrogel causes undesirable effects such as blurred vision, crystal formation on lids and lashes, and irritation.31 Poly(acrylic acid) gel in a nanoscale size may overcome these drawbacks. The present work aims at synthesizing poly(acrylic acid)based nanogel particles as a drug carrier for improving pilocarpine chemical stability, efficacy, and bioavailability. The formation of hydrogel nanoparticles without using organic solvents and surfactants is considered a challenge. Ionizing radiation methods are simple, efficient, and promising for the preparation of biomaterials because they are free of initiators and perhaps other additives. Henke et al. reported an approach that involved irradiation of polyvinylpyrrolidone−poly(acrylic acid) (PVP/PAAc) complexes by pulses of fast electrons in dilute, deoxygenated solutions.32 In this regard, acrylic acid in an aqueous solution of polyvinylpyrrolidone (PVP) as a template polymer was exposed to γ rays to produce chemically cross-linked PVP/PAAc nanogels. Characterization of the PVP/PAAc nanogels is carried out, including morphological structure, pH sensitivity, mucoadhesion, and rheological characteristics. Pilocarpine-loaded nanoparticles were prepared and the in vitro release of pilocarpine from the nanogel particles was evaluated in simulated tear fluid at pH 6 using a dialysis technique.

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loading efficiency(%) = (total amount of drug fed initially − amount of drug in supernatant) × 100/total amount of drug fed initially Particle Size and Zeta Potential Measurements. Particle sizes and zeta potentials of the prepared nanogel particles were measured by the dynamic light scattering (DLS) and electrophoretic light scattering (ELS) techniques, respectively, using a PSS-NICOMP zeta potential/ particle sizer 380ZLS (PSS-NICOMP, Santa Barbara, CA, U.S.A.). For DLS measurements, samples were properly diluted with freshly prepared deionized water until an intensity of 250−350 kHz was achieved. The scattered light intensity was detected at a 90° angle and measurements were run using two 5 min cycles. The volume-weighted hydrodynamic mean diameters were reported to know the size contributing the most volume. The polydispersity index (PDI; the square of the coefficient of variation), which is a measure of the size distribution breadth, was also calculated. For zeta potential measurements, samples were properly diluted with freshly prepared deionized water and then measured at an applied electric field of strength 10 V/ cm using three 30 s cycles for each sample on not less than three independently prepared samples, and their average was recorded. Rheological Measurements. The dynamic viscosity (η′, the real part of the complex viscosity) of the PVP/PAAc nanogel dispersions (at pH 7) was determined using oscillatory squeeze flow via a piezo axial vibrator apparatus in accordance with published methods.33 The sample (∼100 μL) is placed between two circular plates with a diameter of 20 mm. A gap width of 9 μm was selected due to the low viscosities of the solutions. The upper plate is constructed as a gastight lid and is tightly screwed to the base plate. The lower plate is driven through two pairs of piezoelectric actuators. By applying a sinusoidal voltage via a lock-in amplifier, the plate oscillates about a mean position. Two further piezoelectric sensor pairs deliver the

EXPERIMENTAL SECTION

Materials. Polyvinylpyrrolidone (K85−95, Mw = 1300000) and acrylic acid (AAc) were obtained from Acros Organics (Belgium). (+)-Pilocarpine hydrochloride (99%, Scheme 1) was purchased from Alfa Aesar (Germany). Mucin type II obtained from porcine stomach

Scheme 1. Chemical Structure of Pilocarpine HCl

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response signal. We compared a measurement with sample with a measurement of the empty cell and calculated the desired rheological properties from the ratio of the output voltage with and without sample U/U0 and the corresponding phase shift φ−φ0. Measurements were carried out at frequencies ranging from 1 to 1000 Hz at a temperature of 32 ± 0.1 °C. FT-IR Spectrum Analysis. FT-IR spectra of samples were measured as KBr discs by a JASCO FT/IR-6300 spectrometer in the range of 400 to 4000 cm−1. Transmittance Measurements. Transmittance of PVP/PAAc nanogel samples was measured at 500 nm with a JASCO UV/vis spectrophotometer V-560. The medium pH was adjusted by adding few drops of HCl or NaOH solution. Transmission Electron Microscopy Study. Morphology of nanogel particles was observed using transmission electron microscopy (TEM; JEOL JEM-100CX, Japan). Samples were dripped onto a carbon-coated copper grid without staining followed by drying at room temperature. Interaction with Mucin. Two methods were used to assess the mucoadhesive interaction of the prepared PVP/PAAc nanogels with mucin: rheological method and zeta potential method. Rheological Method. A simple rheological method was used to study the mucoadhesive interaction of the prepared PVP/PAAc nanogels.34 An interaction between nanogels and mucin was evaluated by measuring the rheological responses of the nanogel/mucin mixture and the single components (PVP/PAAc nanogel and mucin) using the piezo axial vibrator apparatus, as described earlier. Dried mucin was hydrated with simulated tear fluid (STF, composition: NaCl 0.67 g, NaHCO3 0.20 g, CaCl2·2H2O 0.008 g, and distilled deionized water to 100 g)35 at pH 7 by stirring for 12 h at room temperature, yielding a dispersion of 8% (w/v). The first single-component solution (PVP/ PAAc nanogel/STF) is a mixture containing equal amounts of PVP/ PAAc nanogel dispersion 4% and STF. The second single-component solution (mucin/STF) consists of mucin 8% (w/v), mixed with an equal amount of STF. The PVP/PAAc nanogel/mucin mixture was measured after incubation for 2 h. Dynamic viscosities of PVP/PAAc nanogel/mucin systems, PVP/PAAc nanogel/STF, and mucin/STF were measured at different frequencies. The measurements were performed at 32 ± 0.1 °C. Zeta Potential Method. This method evaluated the influence of the mucin on the zeta potential of the PVP/PAAc nanogels. Nanogels (1.5 w/v %, 3 mL) were incubated at room temperature in aqueous solution of mucin (1 w/v %, 3 mL) under moderate stirring for 1 h. The zeta potentials of the nanogels, mucin, and nanogel/mucin mixtures were determined after a 10-fold dilution with deionized water. In Vitro Release of Pilocarpine. The in vitro release of pilocarpine from the nanogel particles was evaluated in simulated tear fluid at pH 6 through a dialysis membrane (molecular weight cutoff 12000−14000 Da) using a modified apparatus.36 The membrane was first soaked in the dissolution medium for 1 h and then was tied to one end of a specifically designed glass cylinder (open at both ends). A dispersion of drug-loaded particles in STF (5 mg/mL, 2 mL) was placed into this assembly. The cylinder was attached to a stand and suspended in 50 mL of dissolution medium maintained at 32 ± 1 °C and the membrane is approximately 1 cm below the receptor medium surface. The dissolution medium (STF) was stirred with a magnet at 50 rpm. Released pilocarpine was sampled at selected time intervals and determined spectrophotometrically, as described above. The release profile of pilocarpine was compared with a control sample in which pilocarpine was dissolved directly in STF solution and its release profile was measured using the same procedure. All measurements were performed in triplicate and plotted as mean ± SD.

successfully produce nanogel particles with a covalent interior network. The mechanism of nanogel formation by radiation is very complicated, and the main factor responsible for nanogel formation from our point of view is the interpolymer complexation (IPC) via H-bonding between the protondonating poly(acrylic acid) (PAAc), produced in situ, and the proton-accepting PVP. Other contributing factors include the high stability of the produced nanoparticles against aggregation, and the ability of the polymeric components (PVP and PAAc) to cross-link by radiation. PVP/PAAc interpolymer complexes (IPCs) can be efficiently formed only below a critical pH value (around 4) which depends on the molar ratios and molecular weights of the components (Scheme2).37 It is worth noting that Scheme 2. Complexation between PVP and PAAc via HBonding

the nanogel was not obtained when the pH value of the feed solution was raised to more than ∼4.5. Moreover, the pH values of the PVP/PAAc nanogel solutions were slightly higher than those of the corresponding unirradiated feed solutions due to the formation of hydrogen bonding between the nanogel components during radiation processing. In this technique, the major part of radiation is absorbed by water because dilute solutions are used, resulting in water radiolysis. Reactive products of water radiolysis, mainly hydroxyl radicals and H-atoms, can in turn attack AAc or PVP molecules present in the feed solution and accordingly macroradicals are formed (an indirect effect). At the first stage of the irradiation process, chain polymerization of AAc monomer is appeared to be predominant. When the PAAc chains, produced in situ, reaches a critical length, they selfassemble and form IPCs simultaneously with the template PVP molecules. According to Rainaldi et al.,38 it was proposed that the PAAc growing chains of critical length associate with the template PVP by hydrogen bonding and then propagate in contact with the template (pick-up mechanism). AAc may be also grafted onto the PVP molecules when the PVP radicals, formed as a result of hydrogen abstraction, recombine with PAAc radicals or initiate the polymerization of AAc monomer. The resultant hydrophobic complexes aggregate to form nanoparticles of “micelle-like” structure. As no macroscopic precipitation takes place in this process, it could be suggested that the PVP or PAAc chains with less complexed segments in the periphery of the nanoparticles may still be kept solvated and, thus, stabilize the complex aggregates. After most of monomer molecules are polymerized and nanoparticles are formed, radiation is employed mainly in cross-linking of the polymer chains inside the particles (intraparticle cross-linking) producing chemically stable nanogels. In this stage, radiationinduced modification of PVP may play a role in nanoparticle cross-linking. Irradiation of PVP under an air atmosphere induces a variety of polymeric reactive species simultaneously, which leads to oxidation as well as cross-linking due to radical recombination of macroradicals.

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RESULTS AND DISCUSSIONS Preparation of PVP/PAAc Nanogel Particles. γ irradiation of acrylic acid (AAc) monomer in an aqueous solution of polyvinylpyrrolidone (PVP) as a template polymer could 690

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To support the proposed two stages mechanism, the particle size of the prepared nanogel at different doses was measured at pH 7 (at which the PVP/PAAc complex dissociates). It was found that a dose of about 1 kGy is enough to form nanoparticles by IPC between PVP and PAAc chains produced in situ, as revealed from the decrease in transmittance of the feed solution at 500 nm (i.e., the feed solution changes from transparent to opaque one).39 Although nanoparticles are formed, they dissolve at pH 7 because their DLS result is similar to that of corresponding feed solution. This indicated that there is no chemical cross-linking between the chains within the particles prepared at low doses. When radiation doses of 10 to 40 kGy are used, the particle sizes of the prepared nanogels are almost the same when measured at pH 4. Interestingly, these PVP/PAAc particles did not dissolve at pH 7, but instead they swelled. The nanogel particle size at pH 7 decreases as the irradiation dose increases. As a result, at high irradiation doses, the particles became chemically cross-linked. When an aqueous solution of already prepared PAAc was mixed with PVP solution, it gives immediately hydrophobic particles or aggregates. However, AAc monomer gives nothing when mixed with PVP solution. Thus, AAc monomer was first homopolymerized and/or grafted onto PVP when exposed to γ irradiation and then formed IPCs with PVP. Residual AAc monomer and soluble PAAc were determined after nanogel production to ensure that all the amount of AAc in the feed solution is polymerized and involved in nanogel formation. Almost no residual AAc monomer or soluble polymers were detected in supernatant solution of ultracentrifuged nanogel prepared from AAc-rich feed solution. This means that the γ radiation dose used for nanogel preparation (20 kGy) is sufficient to convert the AAc and PVP into PVP/ PAAc insoluble form. However, for nanogel prepared from PVP-rich feed solution unreacted PVP was detected. FTIR spectra for PVP, PAAc, and PVP/PAAc nanogel particles were recorded and it is observed that the CO for PVP and PAAc appears at 1661 and 1720 cm−1, respectively. The PAAc alone has a band at 1720 cm−1 due to the intermolecular hydrogen bonding between the carboxyl groups of PAAc. Once an interpolymer complex has formed, the carbonyl absorption band of PAAc shifted to a higher wavenumber at 1730 cm−1 and that for PVP shifted to a lower wavenumber at 1636 cm−1.40,41 The shifting in the carbonyl absorption bands of PVP and PAAc indicated that a complex is formed between the two components through hydrogen bonding. Controlling the Size of PVP/PAAc Nanogel Particles. To obtain PVP/PAAc nanogels of tunable sizes and capable of entrapping drug efficiently, many factors affecting and controlling the size and cross-linking density of PVP/PAAc nanogel particles have been studied elsewhere in details.39 Among the important factors that affect the size and structure of PVP/PAAc nanoparticles are the PVP/AAc composition and concentration. The feed composition plays an important role in controlling the PVP/PAAc nanogel particle size. An apparent macroscopic PVP gel phase and PAAc soluble polymer were produced when PVP and AAc monomer were exposed individually to 20 kGy at 1.5 w/v % concentration in water. On the other hand, irradiation of a binary PVP/AAc system at different compositions leads to formation of nano or microgels depending on the PVP/AAc molar ratios. Figure 1 shows the effect of feed composition on the PVP/PAAc gel particle size

Figure 1. Effect of feed composition on the particle size of the prepared PVP/PAAc nanogels dispersed in deionized water at pH 4 and 7. Feed conc.: 1.5 w/v %; 20 kGy. Numbers inside parentheses represent polydispersity index (PDI).

measured at low and high pH values using the DLS technique. Synergistic effect on nanoparticle formation by combining both the reactants clearly appears. Nanogel particles of hydrodynamic mean diameters ranging from 80 to 120 nm (measured at low pH) are formed when PVP/AAc feed compositions of molar ratios between 25/75 and 55/45 are used. At these compositions, it is proposed that the complexation between the template PVP and the growing PAAc chains leads to a contraction of the component polymer coils. Further decreases in the PVP/AAc molar ratio result in a sharp increase in the size of the nanogel particles. The higher the AAc content in the feed solution, the higher the amount of AAc diffused to participate in the polymerization at the near surface region of the particles. Subsequently, an increase in the particle size takes place. When the DLS measurements are conducted at pH 7, it is observed that the particle size is higher than that measured at pH 4. In addition, the mean diameter of the nanogel particles increases by increasing the AAc content in the feed solution. At high pH value, the carboxylic acid groups of PAAc chains become ionized resulting in dissociation of H-bonded PVP/ PAAc IPCs and electrostatic repulsion between the ionized PAAc chain segments. This strengthens the water swellability of PVP/PAAc chains and, as a result, the swelling of the PVP/ PAAc nanogel particles increases. Moreover, the higher the content of PAAc in the nanogel particles, the higher the amount of water molecules that penetrates the nanogel particles and, subsequently, the higher the swelling capacity of the particles. The lower values of polydispersity index (PDI) obtained in this study corroborate the observation for relatively monodisperse systems. The values of PDI that are less than (0.2) in the figure reflect the uniformity and monomodal distribution for the particles prepared at different compositions. The average zeta potentials of the nanogel particles, prepared at different compositions and dispersed in deionized water, were determined to evaluate the surface charge and stability of the gel particles (Table 1). The results demonstrated that the particles are negatively charged, indicating the presence of carboxylic groups on their surface. As the amount of AAc increases in the feed solution the zeta potential value slightly increases; the magnitude of zeta potential increases from −18.4 691

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the chain intermolecular recombination, which may lead to the formation of micro- or macrogels is promoted. Thus, the particle size of the prepared PVP/PAAc gels increases. The relatively high PDI at high pH for the particles produced from the lowest and highest feed concentrations may support these suggestions. The yields and nitrogen contents of PVP/PAAc nanoparticles which are prepared from AAc-rich feed compositions were determined. The compositions of nanoparticles (calculated based on their nitrogen contents) are similar with the corresponding feed compositions. Additionally, the yield of PVP/PAAc nanoparticles obtained from the feed solution of high AAc content is high (ca. 95%). This indicated that nanoparticles are formed from both components. Interestingly, the pH induced swelling behavior of the prepared nanogel particles was found to be reversible. This provides an evidence for the chemical “stability” of the formed polymeric architectures. Drug Loading. Pilocarpine was entrapped into the PVP/ PAAc colloidal nanogels by simple mixing of this drug with the aqueous dispersion of the nanogels. Trials were made to load pilocarpine into PVP/PAAc colloidal nanogels at different pHs. Insignificant pilocarpine loading efficiency was recorded at pH 4.5−5. This is evidently due to partial protonation of the carboxylic groups in the PAAc chains (pKa ∼ 4.7) of the nanogels at acidic pH. Meanwhile, it was found that pH 6 is appropriate for pilocarpine loading. At this pH value, both the pilocarpine molecule (pKa = 7.2) and the carboxylic group of the PAAc (around pKa of 4.7) were completely ionized, providing an opportunity to tune the electrostatic binding between the drug and PAAc chains of the nanogels.42 Thus, the loading of pilocarpine into PVP/PAAc colloidal nanogels prepared at different compositions and irradiation doses was carried out at pH 6. It could be seen from Table 2 that pilocarpine loading efficiency into PVP/PAAc colloidal nanogels depends on the

Table 1. Average Zeta Potentials of the Nanogel Particles Prepared at Different Compositions and Dispersed in Deionized Water feed composition PVP/AAc (mol/mol %) 15/85 25/75 35/65 40/60 55/45

zeta potential (mV) −30.8 −24.0 −22.6 −17.1 −18.4

± ± ± ± ±

3.7 1.4 0.3 2.4 1.4

to −30.8 (mV) when the PVP/PAAc composition change from 55/45 to 25/75 (PVP/AAc mol/mol %). the zeta potential values of the nanoparticles prepared at different compositions reflect their surface charge and stability against aggregation. The feed concentration is another important parameter through which the particle size of the prepared PVP/PAAc nanogels can be controlled. At low pH, it can be seen from Figure 2 that the particle size decreases with the increase of

Figure 2. Effect of feed concentration on the particle size of the prepared PVP/PAAc nanogels measured in deionized water at pH 4 and 7. Feed composition: PVP/AAc (35/65 mol/mol %), 20 kGy. Numbers inside parentheses represent polydispersity index (PDI).

Table 2. Loading Efficiency of Pilocarpine into PVP/PAAc Nanogels as a Function of Feed Composition and Irradiation Dosea

feed concentration till reaching about 1 w/v %. Thereafter, any increases in feed solution concentration leads to a limited gradual increase in the average particle size while keeping the feed composition constant. The same behavior is noticed when the size of prepared particles is measured at high pH. As mentioned above, AAc is polymerized and begins to form IPCs with PVP molecules when it reaches a critical length. Thus, at a low feed concentration, it is expected that the length of the produced PAAc chains is not highly enough to complex effectively with PVP molecules. As a result, incompact nanogel structures of relatively high average size are formed. By increasing the feed concentration, the produced PAAc chains become sufficiently long to complex effectively with PVP molecules and small sized nanoparticles are formed to reach a minimum mean diameter at about 1 w/v % feed concentration. Further increases in the feed concentration (higher than 1 w/v %) lead to a significant increase in the viscosities of the prepared PVP/AAc mixtures due to the increase in PVP concentration. This might influence chain flexibility, chain length, and dynamics of PAAc molecules. Consequently, the reorientation ability of PAAc molecules to form IPCs with PVP become slightly limited and nanogel structure of relatively large size is formed. Moreover, as the feed concentration increases,

feed composition (PVP/AAc; mol/mol)

irradiation dose (kGy)

loading efficiency (%)

25/75

20 40 20 40 20 40

25.8 ± 2.6 48.7 ± 3.2 25.4 ± 2.2 47.3 ± 3.3 5.2 ± 3.5 12.0 ± 3.7

35/65 55/45 a

The concentration of the nanogels in water was 5 mg/mL and the drug concentration was 1 mg/mL.

feed composition and the irradiation dose at which the nanogel particles were obtained. AAc-rich nanogels exhibited the highest loading efficiency. These results further reinforce the conclusion that pilocarpine binding to the ionic PVP/PAAc nanogels depended mainly on electrostatic interactions. As the amount of PAAc increases, the chance for the drug to bind electrostatically with the nanogel increases and thus pilocarpine loading efficiency increases. On the other side, irradiation dose at which nanogels were formed greatly affected the drug loading efficiency; the higher the dose the higher the pilocarpine loading efficiency into PVP/PAAc colloid. Nanogels prepared 692

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Figure 3. DLS results for PVP/PAAc nanogels (a1) and pilocarpine-loaded nanogel (b1) at pH 6, and their corresponding TEM images (a2, b2). Feed composition: PVP/AAc (35/65 mol/mol %). Irradiation dose: 20 kGy.

Figure 4. FTIR spectra for pilocarpine HCl, neutralized PVP/PAAc nanogel, and pilocarpine-loaded nanogel.

The amount of loaded pilocarpine into PVP/PAAc nanogels is influenced also by the drug/nanogel weight ratio during the loading process. It was found that the pilocarpine content increased from ∼ 9.5 to ∼ 25 w/w % when the drug/nanogel weight ratio increased from 1/5 to 1/1, respectively, for nanogel particles prepared at 40 kGy using PVP/AAc feed composition of 35/65 (mol/mol %).

at a low irradiation dose possess a low density network structure, resulting in easy diffusion and effusion of the drug.43 Moreover, during nanogel separation by the ultracentrifuge, the diffusion of incorporated pilocarpine molecules from the nanogel particles of high cross-linking density may be lower than that from nanogels of lower cross-linking, since the large centrifugal forces may expel the drug from the nanogel vehicles. 693

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The particle size and morphology of the nanogel particles prepared at 20 kGy using PVP/AAc feed composition of 35/65 (mol/mol %) were investigated by the DLS and TEM techniques before and after drug loading (Figure 3). The hydrodynamic mean diameters of free nanogel and pilocarpine loaded nanogel are about 409 and 345 nm, respectively. This reduction in size was attributed to the partial charge neutralization resulted from the electrostatic interactions between the negative ionized groups of the nanogel and the positively ionized nitrogen of the pilocarpine hydrochloride. This charge neutralization is associated with a reduction in the hydrophilicity of the particles which leads to a decrease in their swelling degree (deswelling) and average size. TEM images showed that the particles loaded with drug seems to be spherical and compact if compared with unloaded ones. This ensures that the overall effect of drug loading produced a less polar environment within the nanogel which produced a more compact nanogel structure.44,45 Moreover, zeta potential measurements provided another evidence of the neutralization of the PAAc segments in the nanogel particles due to the electrostatic binding of pilocarpine.46 It was noticed that upon loading with pilocarpine, the magnitude of the net negative charge of the nanogels was decreased from −46.8 ± 2.1 to −37.3 ± 0.5 mV. The zeta potential values of the nanogels at neutral pH were high. This may reflect the colloidal stability of nanogels which are stable even after storage for more than 6 months. The FTIR spectra of pilocarpine HCl, neutralized PVP/ PAAc nanogel and pilocarpine-loaded nanogel are shown in Figure 4. It can be observed that the FT-IR spectrum of pilocarpine shows characteristic bands at 3082, 3028, 1766, and 1612 cm−1 due to absorptions of quaternary N−CH3, aromatic CH, CO (lactone ring), and aromatic CC, respectively.47 In the PVP/PAAc nanogel spectrum, a characteristic band at 1566 cm−1 appears due to the carboxylate ions of the neutralized nanogel. In addition, there are two overlapping bands at about 1718 and 1661 cm−1 corresponding to CO stretch of nonionized carboxylic groups and N-vinylpyrrolidone ring, respectively. This reveals that not all carboxylic groups are converted into carboxylate ions due to steric hindrance and repulsion forces between ionized groups. The FTIR spectrum of pilocarpine-loaded nanogel shows the carbonyl absorption band of the pilocarpine lactone ring at 1766. Moreover, it can be observed that the carbonyl band of carboxylic groups almost disappears and conversely the carbonyl band of carboxylate ions is enhanced and slightly shifted to a higher frequency at 1574 cm−1. The CO stretch of the N-vinylpyrrolidone ring can also be seen at 1666 cm−1. These differences in the FTIR spectrum of pilocarpine-loaded nanogel in comparison with that of pure nanogel might confirm the electrostatic interaction between the carboxylate ions of the nanogel and the protonated pilocarpine molecules. The mechanism of radiation synthesis of PVP/PAAc nanogels followed by loading with pilocarpine was postulated as shown in Scheme 3. Chemical Stability of Pilocarpine. A problem with the administration of pilocarpine in aqueous eye drops is that the aqueous solution of pilocarpine is subjected to chemical degradation. The main reactions that contribute to its instability are the hydrolysis of the lactone moiety, producing pilocarpic acid.47,48 For this reason, the eye drops are commercially formulated at pH between 4 and 5 in order to obtain an optimal stability during validity time, despite the drug being almost ionized at these values, which can reduce its ocular

Scheme 3. Loading of Pilocarpine into PVP/PAAc Nanogels Prepared by Radiation

absorption.23,24 Thus, the stability of loaded pilocarpine entrapped into nanogel was investigated. FTIR of pilocarpineloaded nanogel stored for 6 months in refrigerator showed pilocarpine characteristic lactone moiety band at 1766 cm−1. This means that the electrostatic interaction between the ternary amine group of pilocarpine drug and carboxylic acid group of PVP/PAAc results in a decrease in the effective number of collisions that occurred between the molecules present in the dispersion. The electrostatic interaction was demonstrated to have a positive effect on drug stability. Transparency of PVP/PAAc Nanogels. To prevent blurred vision, high transparency of an ophthalmic formulation is highly desirable. Therefore, the transmittance at different pH values of the PVP/PAAc nanogel aqueous dispersions loaded with pilocarpine was evaluated at 500 nm (Figure 5). It can be

Figure 5. Dependence of transmittance (at 500 nm) of the PVP/PAAc nanogels on the pH value (inset is photos for the prepared nanogels (1.5%) at pH 4 and 7). Feed composition: PVP/AAc (35/65 mol/mol %); 20 kGy.

observed that the transmittance of the PVP/PAAc nanogel dispersions increased generally with the increase of the pH value to reach a maximum at pH ∼ 5.8; thereafter, any increases in pH leads to insignificant increases in transmittance. This can be attributed to the pH-induced phase transition phenomenon of interpolymer complexes.49 At low pH, the particles have a compact structure due to the hydrophobic interactions resulting from the involvement of hydrophilic groups in direct hydrogen bonding. As the pH increases, the strong hydrogen bonding is weakened and the water-solubility of the chain segments within 694

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the nanogel particles is strengthened resulting in more transparent nanogel dispersion. Another factor affect the % transparency of the nanogel dispersion at neutral pH is irradiation dose used for nanogel preparation. As the irradiation dose increases from 20 to 40 kGy, the % transparency of nanogel decreases from ∼46.5 to ∼5.0. At a higher irradiation dose, the produced nanogel particles have a polymer network structure of high cross-linking density (more compact and hydrophobic); the higher the cross-linking density, the lower the transmittance of nanogel particle. It was reported that ideal characteristics of ocular drug delivery systems50,51 are good corneal penetration, prolonged contact time with corneal tissue, simplicity of installation for the patient, and nonirritative and comfortable form (the viscous solution should not provoke lacrimation and reflex blinking). Therefore, rheological, irritating, and mucoadhesion properties of PVP/PAAc nanogels should be investigated to show to what extent the prepared nanogel could be used as an ocular delivery matrix. Irritation Effect of PVP/PAAc Nanogels. The possibility of eye irritation due to administration of PVP/PAAc nanogels was evaluated in rabbits. An ophthalmologist examined the rabbits under investigation for ocular lesions. Ocular irritation symptoms such as tearing, redness, inflammation or swelling were not observed after nanogel administration. Corneal or conjunctival epithelial defects were not detected when fluorescein staining was used. PVP/PAAc nanogels were also tested for treatment of eye dryness in rabbits, which induced by 1% atropine sulfate solution. It was noted that the nanogels had no irritant effect on the rabbits’ eyes. In contrast, PVP/PAAc nanogels reversed the inflammatory signs resulting from dryness (meiosis, photophobia and conjunctival congestion) after treatment for 7 days twice daily (data not published). Rheological Properties of PVP/PAAc Nanogels. Many factors can control the viscosity of PVP/PAAc nanogels such as environmental pH, and preparation conditions (composition and irradiation dose). Environmental pH affects greatly the viscosity of the prepared nanogels. At low pH (less than pKa of PAAc, 4.7), the viscosities of PVP/PAAc nanogels prepared at different irradiation doses and compositions are comparable and close to water viscosity. As described previously, the lower viscosity at low pH is due to the strong hydrogen bonding among the chain segments of the nanogel particles. The nanogel in this form is expected to be unsuitable for ophthalmic uses due to its low viscosity and acidity properties. In order to utilize PVP/PAAc nanogels as carrier for ocular drugs their environmental pH was raised to improve their viscosities and change them to neutral form. Viscosities of PVP/PAAc nanogels prepared at different compositions and irradiated with 20 and 40 kGy were measured at pH 7. Figure 6 shows changes of viscosities as a function of frequency for the nanogel dispersions at a concentration of 2 w/v %. The viscosity of the nanogel dispersions is correlated with the content of AAc in the nanogel as well as the crosslinking density. At the same frequency, the viscosity increases with a larger amount of AAc and a lower cross-linking density in the nanogel. At a high content of AAc and low cross-linking density, the enhanced osmotic pressure exerted by the counterions trapped inside the polymeric network by the large number of charged carboxylate groups causes an increase in the effective volume occupied by the particles. Such an increase in volume essentially decreases the interparticle

Figure 6. Viscosities as a function of frequency for the PVP/PAAc nanogels prepared from different PVP/AAc compositions at 20 and 40 kGy and dispersed in water.

distance, where the interaction forces between the swollen particles increase, resulting in the increase of viscosity.52 In addition, the nanogel dispersions display a pseudoplastic behavior. This can be ascribed to the hysteresis of the polymer chain motion in comparison with the frequency supplied by the test machine, resulting in a decrease in the interaction of the polymer chains with the surrounding water.53 The lower viscosities of these PAAc-based nanogels could be useful in promoting good retention and stability on the eye without significant discomfort to the patient. To apply the PVP/PAAc nanogels’ solutions topically into the eye, nanogel apparent viscosities should be measured in a simulated tear fluid. Figure 7 shows the dynamic viscosities of

Figure 7. Viscosities as a function of frequency for the PVP/PAAc nanogels prepared from different PVP/AAc compositions at 20 and 40 kGy and dispersed in a simulated tear fluid.

PVP/PAAc nanogels prepared at different PVP/AAc compositions and dispersed in a simulated tear fluid as a function of frequency. It could be observed that the viscosities of the nanogels dispersed in simulated tear fluid are lower than those dispersed in water. It is well-known that salts like NaCl can affect the viscosity of polyelectrolytes. The salts would screen the electrostatic repulsion between the ionized PAAc chain segments, promoting the collapse of the chains.54 Therefore, 695

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Zeta potential measurements were also used as another method to evaluate the interaction of PVP/PAAc nanogels with mucin dispersion before and after loading with pilocarpine.56 In the test, mucin was dispersed in water at a concentration of 1% and the pH was raised to 6 using drops of NaHCO3 solution. Mucin dispersion (1 w/v %, 3 mL) was then mixed with (1.5 w/v %, 3 mL) nanogel solution. It was expected that the surface charge of mucin might be changed by the adhesion of the polymeric nanoparticles if they have a mucoadhesive property. The occurrence of such change was detected by measuring the zeta potential. The zeta potential value of the mucin alone at pH 6 is −15.5 ± 0.7 mV, which is in a good agreement with the data previously reported by Takeuchi et al.56 The negative charge of mucin particles can be explained by the presence of 1% of bound sialic acid. As it can be seen from Table 3, when the mucin dispersions (of particle size in micrometers) were mixed with the solutions of both drug-loaded and free nanogels, the zeta potential of the mucin changed to a higher negative value due to the negative charge of the nanogels. The zeta potential value of mucin−PVP/PAAc nanogel prepared at 20 kGy is slightly higher than that of mucin−PVP/PAAc nanogel prepared at 40 kGy. This might be attributed to the high crosslinking yield of PVP/PAAc nanogel prepared at 40 kGy that restricted the mobility of chains and reduced their interaction with mucin. The obtained results indicated that PVP/PAAc nanogels had a high affinity to interact with mucin and cover its surfaces. This strongly supported mucoadhesive property of PVP/PAAc nanoparticulate gels. In Vitro Release of Pilocarpine. The most common types of glaucoma are chronic and require lifelong treatment. With simple eye drops, it is not possible to maintain therapeutic concentration for a prolonged time, and frequent dosing often leads to compliance failures as well as an increased risk of side effects. Reducing the dosing frequency should improve patient compliance, which is necessary in glaucoma therapy. A sustained pilocarpine release can be a solution to overcome this problem. Thus, pilocarpine was loaded into PVP/PAAc nanogel and its release was evaluated at 32 ± 1 °C in simulated tear fluid (STF, pH 6). The release profile of pilocarpine-loaded nanogel (with a loading efficiency of 47%) was compared with that of a control sample in which pilocarpine was dissolved directly in STF at pH 6 (Figure 9). The release rate of pilocarpine from the control sample through the dialysis membrane was relatively very fast if compared with that from the pilocarpine-loaded nanogel sample. It was found that about 35% of the pilocarpine was released from the control sample into the medium within 15 min, and most of the drug was released within 6 h. However, in case of the pilocarpine-loaded nanogel, only about 10% of the drug was released within the first 15 min, and then about 55% of the drug was released within 6 h. The interaction between pilocarpine and PVP/PAAc nanogel resulted in delaying the release of pilocarpine in STF solution.

the interactions of nanogel−nanogel particles and nanogel particles−water decrease, resulting in the decrease of viscosity. Interaction with Mucin (Mucoadhesion). The interaction between drug carrier and precorneal mucin (mucoadhesion) plays an important role in precorneal drug absorption and depends on the functionality and charges of carriers.11 Therefore, according to Hassan and Gallo,34 rheological properties (viscosity) of PVP/PAAc nanogel particles prepared at different irradiation doses and their mixtures with commercial porcine stomach mucin as well as pure mucin were investigated (Figure 8). Rossi et al.55 justified the use of

Figure 8. Changes of viscosity as a function of frequency for aqueous dispersions of nanogel/STF, nanogel/mucin, and mucin/STF. Nanogel feed composition: 35/65 PVP/AAc mol/mol %. Irr. Dose: 20 and 40 kGy.

commercial mucins by the lower batch-to-batch variability shown by commercial samples with respect to those freshly prepared. In general, for all components and their mixtures investigated here, as the frequency increases, the solution apparent viscosity decreases. An interaction between the nanogels and mucin could be seen as a synergistic effect in the rheological properties of the nanogel/mucin mixtures if compared with those of the corresponding individual components (PVP/PAAc nanogel particles and mucin) due to either physical entanglements or secondary bonds between them. The rheological response of the PVP/PAAc−mucin mixture is larger than the sum of the rheological response of the individual components. Furthermore, the rheological response of the mucin−PVP/PAAc nanogel prepared at 20 kGy is higher than that of mucin−PVP/AAc nanogel prepared at 40kGy due to the viscosity difference. The improvement in viscosity of nanogel/mucin indicated the mucoadhesion properties of PVP/ PAAc nanogel particles.

Table 3. Zeta Potentials (mV; Measured at pH 6) of Free Nanogel, Pilocarpine-Loaded Nanogel and Their Corresponding Mixtures with Mucin at Weight Ratio of 3:2 dose

nanogela

nanogel/drugb

nanogel/mucin

nanogel/drug/mucin

20 kGy 40 kGy

−46.8 ± 2.1 −45.2 ± 2.5

−37.3 ± 0.5 −32.8 ± 0.6

−34.1 ± 0.2 −31.0 ± 2.6

−31.5 ± 1.5 −29.1 ± 3.7

Feed composition: 35/65 mol/mol % PVP/AAc (mucin zeta potential = −15.5 ± 0.7 mV). bLoading efficiency is about 25 and 48% for a nanogel prepared at 20 and 40 kGy, respectively. a

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the International Atomic Energy Agency (IAEA) regular budget fund under Research Contract No. 15470/R1.

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REFERENCES

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Figure 9. Cumulative amount of pilocarpine released as a function of time at pH 6 from control (aqueous pilocarpine solution) and loaded PVP/PAAc nanogel dispersion prepared from PVP/AAc feed composition of 35/65 (mol/mol) at 40 kGy.

This interaction is not so strong because of the conceptual low stability of the forming complex species between the COO− of the nanogel and the protonated pilocarpine molecule, which allows the drug to be released slowly from the nanoparticle matrix. These results indicated that the PVP/PAAc nanogel particles are appropriate for pilocarpine delivery. Moreover, these nanoparicles are expected to increase the contact time of pilocarpine with the eye surface and might promote or facilitate transfer of pilocarpine molecules from the tear phase into the eye tissue.

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CONCLUSION Biomedical PVP/PAAc nanogels were prepared successfully using γ radiation. Nanogel particle size was controlled by adjusting radiation dose and PVP/AAc feed concentration and molar ratio. Particles of hydrodynamic mean diameters ranging from 80 to 120 nm (measured at low pH) are formed when PVP/AAc feed solutions of molar ratios between 25/75 and 55/45 at a total concentration of 1.5 w/v % are used. The nanogel particle size was significantly affected by changing the external conditions, like medium pH. Rheological analysis indicates that the nanogels dispersed in tear fluid possess low viscosities and pseudoplastic behavior and could be used as a carrier for ocular delivery without causing blurred vision or patient compliance failure. In addition, mucoadhesive interactions between the nanogels and mucin were determined in terms of viscosity and zeta potential changes and strongly supported mucoadhesive property of PVP/PAAc nanoparticulate gels. Furthermore, pilocarpine loading efficiency depends on the AAc content as well as the cross-linking density of nanoparticles. AAc-rich nanogels of high cross-linking yield exhibited the highest loading efficiency. Pilocarpine-loaded nanogels showed a slow release if compared with pilocarpine solution; only about 55% of the drug was released within 6 h. Nonirritating, mucoadhesive, and rheological properties of PVP/PAAc nanogels as well as their pilocarpine slow release qualify such nanomatrices for possible uses as ocular drug carriers. In conclusion, this study clearly demonstrated the promising potential of PVP/PAAc nanoparticles for multiplying the therapeutic effect of pilocarpine delivery with enhanced bioavailability response. 697

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