Feasibility Investigation of Cellulose Polymers for Mucoadhesive

Article pubs.acs.org/molecularpharmaceutics

Feasibility Investigation of Cellulose Polymers for Mucoadhesive Nasal Drug Delivery Applications Kellisa Hansen,†,⊥ Gwangseong Kim,†,⊥ Kashappa-Goud H. Desai,†,⊥ Hiren Patel,† Karl F. Olsen,† Jaime Curtis-Fisk,§ Elizabeth Tocce,∥ Susan Jordan,§ and Steven P. Schwendeman*,†,‡ †

Department of Pharmaceutical Sciences and the Biointerfaces Institute, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, Michigan 48109, United States ‡ Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States § Formulation Science, ∥Dow Pharma and Food Solutions, The Dow Chemical Company, Midland, Michigan 48674, United States S Supporting Information *

ABSTRACT: The feasibility of various cellulose polymer derivatives, including methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), sodium-carboxymethylcellulose (sodium-CMC), and cationic-hydroxyethylcellulose (cationic-HEC), for use as an excipient to enhance drug delivery in nasal spray formulations was investigated. Three main parameters for evaluating the polymers in nasal drug delivery applications include rheology, ciliary beat frequency (CBF), and permeation across nasal tissue. Reversible thermally induced viscosity enhancement was observed at near nasal physiological temperature when cellulose derivatives were combined with an additional excipient, poly(vinyl caprolactam)−poly(vinyl acetate)−poly(ethylene glycol) graft copolymer (PVCL−PVA− PEG). Cationic-HEC was shown to enhance acyclovir permeation across the nasal mucosa. None of the tested cellulosic polymers caused any adverse effects on porcine nasal tissues and cells, as assessed by alterations in CBF. Upon an increase in polymer concentration, a reduction in CBF was observed when ciliated cells were immersed in the polymer solution, and this decrease returned to baseline when the polymer was removed. While each cellulose derivative exhibited unique advantages for nasal drug delivery applications, none stood out on their own to improve more than one of the performance characteristics examined. Hence, these data may be useful for the development of new cellulose derivatives in nasal drug formulations. KEYWORDS: nasal drug delivery, cellulose, mucoadhesion, permeation enhancement, acyclovir, rheology, thermogelation

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the dosage form via mucoadhesive polymers,1,2,7,9,11,12 (b) the use of permeation-enhancing entities to improve absorption of drug molecules,1,2,7,9,11−13 and (c) coincorporation of enzyme inhibitors to prevent undesired drug degradation.1,2,7 Many different varieties of polymeric excipients have been used to overcome these barriers, each with a specific approach to enhance the performance of the drug delivery system. Chitosan is a well-studied polymer used to increase retention time in the nasal cavity by a bioadhesive mechanism.14 Mucoadhesion has also been achieved by polymeric excipients designed to be sensitive to specific environmental conditions.2 Of particular interest in nasal drug delivery are polymers sensitive to temperature15,16 and pH.17−19 Rheological characterization is often used to evaluate these in situ gelling systems. There are a large number of excipients that have been extensively studied for use as permeation enhancers, including surfactants, bile

INTRODUCTION Nasal mucosa has been recognized as an attractive site for local and systemic delivery of numerous small and large molecule drugs. Nasal delivery has several key advantages, as it (a) provides a desirable alternative to parenteral administration since it is amenable to self-medication and is virtually painless,1,2,4−7 (b) does not require sterile techniques,6 (c) provides rapid drug absorption due to the extensive vasculature and highly permeable structure within the nasal membranes,4−7 (d) avoids the loss of parent drug due to first-pass metabolism in the liver and presystemic elimination in the gastrointestinal tract,1,3−7 (e) may exhibit reduced side effects because it can provide equal drug efficacy at lower doses,1,5,7 and (f) can provide a quick onset of action.1,3,5−7 Despite these unique and beneficial features, nasal drug delivery has three key limitations, including (a) rapid mucociliary clearance, limiting the residence time of the drug product at the site of absorption,2,6−10 (b) local enzymatic degradation of drugs,2,7,8,10 and (c) poor permeability to large and hydrophilic drugs across nasal mucosa.2,7 The most commonly studied strategies to overcome these aforementioned limitations are (a) increasing the local residence time of © 2015 American Chemical Society

Special Issue: Advances in Respiratory and Nasal Drug Delivery Received: Revised: Accepted: Published: 2732

April 4, 2015 June 4, 2015 June 22, 2015 June 22, 2015 DOI: 10.1021/acs.molpharmaceut.5b00264 Mol. Pharmaceutics 2015, 12, 2732−2741

Article

Molecular Pharmaceutics

were received as gift samples from The Dow Chemical Company (Midland, MI, USA). Poly(vinyl caprolactam)− poly(vinyl acetate)−poly(ethylene glycol) graft copolymer (Soluplus) was received as a gift sample from BASF (Florham Park, NJ, USA). Acetonitrile, Krebs-Ringer bicarbonate (KRB) buffer, and acyclovir were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Prigrow I cell medium was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). Porcine nasal tissues were obtained from the University of Michigan Health System Extracorporeal Membrane Oxygenation (ECMO) and Animal Surgery Operating Rooms (ASOR) Laboratories (Ann Arbor, MI, USA) and from Milligan’s Northwest Market (Jackson, MI, USA). Preparation of Polymer Solutions. The stock polymer solutions of methylcellulose (MC, METHOCEL A4M) and hydroxypropylmethylcellulose (HPMC, METHOCEL K4M) were prepared by a hot and cold technique.24 Briefly, required quantities of polymers (1.5% w/w) were added to doubledistilled water (ddH2O) previously maintained at 80 °C under constant stirring with an overhead mixer at 1000 rpm. The polymer−water mixtures were continuously stirred for 10 min at 80 °C and then at 4−8 °C for 20 min. Sodiumcarboxymethylcellulose (sodium-CMC, WALOCEL CRT 1000 PA) solution was prepared by adding the required quantity of polymer (3% w/w) to ddH2O and stirring the mixture for 6−8 h at room temperature. Cationic-hydroxyethylcellulose (cationic-HEC, UCARE JR 400) was prepared in a two-step procedure. First, the polymer was dissolved in ddH2O (3% w/w) at room temperature until the solution appeared transparent (approximately 1 h); then, it was gradually heated to 65 °C and maintained for 1 h stirring at 1000 rpm. All polymer solutions were then hydrated at 4 °C for 24 h to facilitate sufficient hydration of polymers and to remove air bubbles. Polymer solution concentrations were prepared by diluting with ddH2O. Stock solutions of PVCL−PVA−PEG were prepared by adding a desired quantity of polymer to ddH2O at room temperature and allowing the mixture to continuously stir on a hot plate with a small indentation/vortex for 48 h. Solutions containing more than one polymer component were prepared by adding the desired quantities of stock polymer solutions to a vial and adding sufficient ddH2O to achieve the desired polymer concentrations. Rheological Measurements. The rheological behavior of cellulose derivatives was studied by measuring their viscosity using a rheometer (AR-G2 rheometer, TA Instruments). The geometry used was a 6 cm stainless steel cone and plate with a truncation gap of 21 μm. The measurements were performed under oscillated temperature ramping mode.25 Briefly, approximately 1.2 mL of the polymer solution was added onto the plate and the polymer solution was held at 10 °C for 5 min. Then, the temperature was ramped from 10 to 70 °C at a heating rate of 1 °C/min. Each measurement was conducted in the linear viscoelastic range, as determined by a strain sweep. An oscillation frequency of 1 Hz was applied. Each temperature sweep was conducted in triplicate using freshly prepared stock solutions with representative curves reported. Inflection point temperatures were calculated using proprietary software provided by The Dow Chemical Company and are reported as the average of three temperature sweeps. Tissue Harvesting. After the animals had been euthanized by barbiturate or potassium chloride overdose, the porcine nose was amputated and divided into two parts by cutting along the

salts, cyclodextrins, phospholipids, cationic polymers, lipids, and other miscellaneous systems.20 Other polymeric excipients that have been explored, previously, in nasal drug delivery applications are celluloses. Celluloses are polysaccharides containing 8000−10000 glucose units linked by β-1,4 glucosidic bonds that have uses in a number of pharmaceutical applications. Cellulosic derivatives were suggested to be potential candidate excipients for developing intranasal drug formulations based on their unique physical and chemical properties that can be particularly relevant to nasal drug delivery, including biocompatibility in humans, ease of formulation processing, high availability, and cost effectiveness. The hydroxyl groups present on the glucose units of celluloses are amenable to chemical modifications. Depending on the type of substitution at the hydroxyl position, there are many different types of cellulose derivatives available, such as methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), sodium-carboxymethylcellulose (sodium-CMC), and cationic-hydroxyethylcellulose (cationic-HEC). Significant work has been performed studying the use of some of these derivatives as excipients in nasal drug formulations to enhance delivery and performance. Sodium-CMC has been studied as an intranasal drug carrier in a dry powder formulation to assist in mucoadhesion.21 Water-soluble cationic-HEC has also been shown to possess mucoadhesive properties due to electrostatic interactions between the positively charged polymer chains and negatively charged mucin molecules that are significant for controlled drug delivery systems.22 The long-term objective of our research is to explore different nasal formulations based on cellulose derivatives to aid in intranasal delivery of small and large therapeutic molecules. In this study, we selected four cellulosic derivatives, MC (METHOCEL A4M), HPMC (METHOCEL K4M), sodium-CMC (WALOCEL CRT 1000 PA), and cationic-HEC (UCARE JR 400) (all provided by Dow Chemical Company in premium grade), to investigate their physicochemical properties for nasal delivery preformulation characterization and to use the information gained in these studies to help guide the development of more tailored cellulose derivatives in the future. In addition, Soluplus (BASF), a poly(vinyl caprolactam)−poly(vinyl acetate)−poly(ethylene glycol) graft copolymer (PVCL−PVA−PEG) with interesting drug solubilization properties due to its amphiphilic structure,23 was also blended as an additional excipient with the above polymers in order to improve the drug’s solubility. Characterization of the cellulosics, as described here, included (1) polymer rheology to determine whether any of the dissolved polymers exhibit sufficient viscoelastic behavior to assist in prolonging the residence time, (2) ciliary beat frequency to determine whether any polymer can affect the ciliary beating to delay the mucociliary clearance and if the polymer may have an adverse effect on cells or tissues, and (3) drug permeation across excised nasal mucosa to determine whether the polymers affect the permeation rate. The preformulation polymer characterization strategy described below is designed to aid in the fundamental understanding of the cellulose excipients pertaining to their future formulation in nasal delivery dosage forms.

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MATERIALS AND METHODS Materials. Methylcellulose A4M (METHOCEL A4M), hydroxypropyl methylcellulose K4M (METHOCEL K4M), sodium-carboxymethylcellulose (WALOCEL CRT 1000 PA), and cationic-hydroxyethylcellulose (UCARE Polymer JR 400) 2733

DOI: 10.1021/acs.molpharmaceut.5b00264 Mol. Pharmaceutics 2015, 12, 2732−2741

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Figure 1. Basic rheological properties of four cellulosic polymers over gradual temperature ramping. (a) Representative viscosity profiles of 0.1% cellulosic polymer solutions and (b) representative profiles of 0.1% cellulosic polymer with 5% PVCL−PVA−PEG solutions.

Inc., Santa Clara, CA, USA). The CBF was then counted visually by playing the recorded video at a slower speed (8 frames/second). The CBF values of five different cell populations were measured for each specimen. Ex Vivo Permeation of Acyclovir across Porcine Nasal Mucosa. Ex vivo permeation of an active pharmaceutical ingredient (API), acyclovir, across porcine nasal mucosa was conducted using vertical Franz diffusion cells (PermeGear, Inc., Hellertown, PA, USA) with water-jacketed donor and receiver chamber volumes of 1 and 8 mL, respectively. The diffusional interface was a spherical shape with an exposed diameter of 1.2 cm and area of 1.1 cm2. After harvesting the nasal tissue (see Tissue Harvesting), the epithelium was separated from the underlying connective tissue with forceps and mounted between the donor and receiver chambers. One milliliter of acyclovir (2 mg/mL) or acyclovir (2 mg/mL) + cellulose derivative (0.1% w/w) solution was added to donor chambers. The receiver chamber was filled with 8 mL of KRB buffer and maintained at 35 °C by circulating the water with a thermostatically controlled water bath. The receiver chamber medium was stirred by magnetic stir bar. At 15 min intervals, the entire medium of the receiver chamber was withdrawn and immediately replaced with prewarmed (at 35 °C) fresh medium. Acyclovir content in the donor and receiver chambers was quantified by high-performance liquid chromatography (HPLC). The permeability coefficient of acyclovir across porcine nasal mucosa was then calculated by

nasal septum. The posterior septum was used for the measurement of ciliary beat frequency. The nasal tissues were separated from the turbinate cartilage and kept hydrated with KRB. The excised nasal tissue was then used to measure drug permeation across nasal tissue. The time taken to sacrifice animals and remove the nasal tissue was less than 1.5 h. All studies were conducted immediately after removal of nasal tissues. Measurement of Ciliary Beat Frequency (CBF). The measurement of CBF in the presence and absence of cellulosic derivatives was performed using nasal epithelial cells from excised porcine nasal tissue. CBF measurements were made in one of two ways. CBF was determined using an adapted method previously described by Clary-Meinesz et al.26 Briefly, to determine CBF in the presence of cellulosic derivatives, epithelial cells were harvested by brushing the excised porcine nasal mucosa with a disposable cytological brush. Specimens were resuspended in a combination of cell media (Prigrow I cell medium) and cellulosic derivative to achieve the desired polymer content; then, the samples were equilibrated at 35 °C until the CBF measurement was recorded. To determine CBF after exposure and subsequent removal of cellulosic derivatives, sections of nasal septal tissue (approximately 10 × 10 mm2) were covered with approximately 100 μL of cellulosic derivative at specified concentrations and allowed to equilibrate for 20 min. Cellulosic derivatives were subsequently removed by washing with warmed (35 °C) KRB, and epithelial cells were harvested by brushing the excised porcine nasal mucosa with a disposable cytological brush, resuspended in cell media (Prigrow I cell medium), and equilibrated at 35 °C until the CBF measurement was recorded. To measure CBF, 200 μL of the cell suspension was placed in an 8-well chambered microscope coverglass (Lab-Tek chamber slide, Fisher Scientific), and ciliated cells were examined with an inverted optical microscope (Olympus CK2, Center Valley, PA, USA) using 40× magnification. A temperature controlled microscopy stage (Bioscience Tools, San Diego, US) was used to maintain the cell suspension at 35 °C to mimic in vivo nasal conditions. The beating of ciliated cells was captured by a high-speed camera (30 frames/second) connected to a computer equipped with SeqSnap PCVision, v1.0, software (Imaging Technology,

Peff (cm/s) =

dC VR dt AC D

where Peff = permeability coefficient, dC/dt = rate of change in drug concentration, VR = volume in receiver chamber, A = permeation area, and CD = initial drug concentration in donor chamber. Each permeation study was completed, minimally, in triplicate. Acyclovir HPLC Assay. All HPLC assays were performed with a Waters 2695 Alliance System (Milford, MA, USA) consisting of a 2996 photodiode array detector and a personal computer with Empower 2 software. A Nova-Pak C18 column (4 μm, 150 mm × 3.9 mm) was used. Isocratic elution with 2734


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Table 1. Gelation Temperatures of Aqueous Solutions of Cellulosic Derivatives in the Presence and Absence of PVCL−PVA− PEG observations of increases in viscosity (physiological range, 30−37 °C) cellulosic derivative MC

cellulosic derivative concentration

gelation temperatureb

complex viscosity, |η*|

gelation temperatureb

complex viscosity, |η*|

(w/w %)

(w/w %)

(°C)

(×10−3 Pa s)

(°C)

(×10−3 Pa s)

0

0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25

5 HPMC

0 5

sodium-CMC

0 5

cationic-HEC

0 5

34.8 (0.5)a 30.7 (0.9)

8.34 (0.93) 8.33 (0.83)

36.0 (0.1) 31.9 (0.4)

12.88 (2.43) 10.4 (0.84)

36.9 (0.4) 36.2 (0.7)

17.97 (2.49) 33.15 (15.78)

36.0 (0.4) 35.7 (0.3) 36.3 (0.6)

5 a

(>37 °C)

PVCL−PVA−PEG Concentration

10.42 (2.45) 12.84 (0.62) 5.64 (0.73)

58.2 64.9 63.8 62.4 61.4 62.4 64.8 67.4 62.7 59.7

(6.0) (1.4) (1.4) (0.1) (2.9) (1.8) (1.3) (2.1) (4.4) (5.8)

67.00 430.38 56.54 41.58 99.31 45.75 112.72 15.59 48.25 91.28

(19.63) (345.60) (46.08) (22.20) (71.20) (22.01) (87.09) (1.72) (31.98) (69.75)

65.7 (2.4) 57.9 (4.7)

35.8 (27.07) 15.04 (4.39)

65.1 (1.3) 67.2 (0.2)

80.40 (20.38) 183.56 (92.31)

Number in parentheses represents the standard error of the mean; n = 3. bAs determined by the inflection point of the storage modulus, G′.

Figure 2. Representative rheological profiles of each cellulose polymer (0.1 and 0.25% w/w) with and without PVCL−PVA−PEG (5% w/w) at 10− 70 °C: (a) MC, (b) HPMC, (c) sodium-CMC, and (d) cationic-HEC.

ddH2O was employed at a flow rate of 1.0 mL/min, and the detection wavelength was set at 254 nm.27

Histological Examination. A portion of nasal tissue samples from the 2 h permeation studies and unexposed 2735


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enhancement was observed at 34.8 °C, slightly lower than that of the 5% PVCL−PVA−PEG solution alone, 36.3 °C. As the concentration of MC increased to 0.25%, the onset temperature was even further reduced to 30.7 °C (Figure 2a). A similar phenomenon was observed for HPMC (Figure 2b). The mixture of 5% PVCL−PVA−PEG with 0.1% sodium-CMC showed a significant increase in viscosity, and as the concentration of sodium-CMC increased, so did the viscosity enhancement. However, it did not show any significant change in the onset temperature (which was consistently at approximately 36 °C, Figure 2c). Cationic-HEC exhibited a similar enhancement in viscosity as that of sodium-CMC, but it did so to a lesser extent. The gelation temperature remained at a constant 36 °C. The increase in cationic-HEC concentration did not significantly affect the enhancement of viscosity, as was observed for MC and HPMC, but the maximal viscosity reached was not reduced (Figure 2d). The polymer mixtures were further analyzed by comparing the viscosity of the solutions at their gelation temperatures, as determined by the inflection point of the storage modulus, G′. From this analysis, it was observed that mixtures of MC and PVCL−PVA−PEG showed no significant enhancement in viscosity as compared to that of either polymer solution alone, aside from an additive affect (Figure 3). A similar observation

tissues for control were fixed in buffered 10% formalin and embedded in paraffin wax for hematoxylin and eosin (H&E) staining. Then, 5 μm sections (Leica 2235 rotary paraffin microtome) were placed on microscope slides, deparaffinized using xylene, and rehydrated using ethanol solutions in a gradient of 80% up to 100% and distilled water. The tissue slices were placed in 0.7% w/w hematoxylin solution and rinsed twice in acid ethanol (0.1 N HCl in 95% ethanol) to remove the excess stain. Subsequently, the tissue slices were placed in 0.1% w/w eosin solution and dehydrated using solutions of ethanol in a gradient of 80% up to 100% and then xylene. Light microscopy was then performed using an Olympus BX51 microscope (Olympus, Tokyo, Japan) at 40× magnification. Images of the tissue sections were captured using a fitted camera (Olympus DP70 digital camera, Tokyo, Japan), and software (Olympus DP controller, Tokyo, Japan) for general morphological examination and comparison was used to evaluate the images.

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RESULTS Rheology of Selected Cellulosic Polymer Derivatives. The viscosity profile for the four types of cellulosic polymers (MC, HPMC, sodium-CMC, and cationic-HEC) at 0.1% concentration during temperature ramping from 10 to 70 °C was monitored at a 1 °C/min heating rate. All four polymers exhibited an initial gradual decrease in viscosity over the temperature ramp. At temperatures above 50 °C, MC, HPMC, and cationic-HEC exhibited a sharp rise in viscosity to various maximal values. Sodium-CMC also exhibited a minor increase in viscosity near the same temperature, as shown in Figure 1a. The gelation temperatures for all four cellulosic polymers were approximately 60 °C, as determined by the inflection point of the storage modulus, G′ (Table 1). This viscosity enhancement indicated a potential reverse thermogelation capability of METHOCEL derivatives (including MC and HPMC) and has been previously known.24 However, this thermosensitive gelation behavior occurs at temperatures outside those desired for nasal drug delivery applications. In the physiological temperature range of the nasal cavity, all four polymers showed only slightly higher viscosity than that of aqueous solution. The introduction of a polymeric solubilizer, PVCL−PVA− PEG, to improve the solubility of acyclovir, induced an elevation in viscosity near temperatures relevant to the nasal cavity for all four polymers. As summarized in Figure 1b, all four polymer solutions exhibited an increase in viscosity near 40 °C, by a factor of 10−30. MC and HPMC still exhibited their own increase in viscosity around 60 °C in addition to the event near 35 °C, confirming that the viscosity enhancement at near body temperature was induced by PVCL−PVA−PEG. It appears that the viscosity profile of PVCL−PVA−PEG dominated any behavior that can be attributed to sodiumCMC and cationic-HEC. The interactions between MC, HPMC, sodium-CMC, and cationic-HEC and PVCL−PVA-PEG were further investigated to examine the viscosity enhancement near 40 °C in more detail. The thermogelation behavior was monitored by varying the concentration of cellulose polymers (0.1 and 0.25%). These results are shown in Figure 2, and the gelation temperatures are summarized in Table 1. As mentioned, 5% PVCL−PVA−PEG alone exhibited a slight viscosity increase starting at 35 °C. The mixture of 5% PVCL−PVA−PEG and 0.1% MC showed a small degree of enhancement in viscosity, and the onset temperature of this

Figure 3. Complex viscosities, |η*|, of cellulosic derivatives and PVCL−PVA−PEG solutions at the gelation temperature, as determined by the inflection point of the storage modulus, G′.

was made for HPMC and cationic-HEC. The mixtures of PVCL−PVA−PEG and 0.1% sodium-CMC, alternatively, showed a greater viscosity beyond what can be attributed to an additive occurrence. The mixture of PVCL−PVA−PEG and 0.25% sodium-CMC was inconclusive. Ciliary Beat Frequency. The ciliary beat frequency of primary nasal epithelial cells observed in the absence of excipients was determined to be 11−12 Hz. When submersed in a solution of stock cellulosic polymer solution diluted to the desired concentration with cell culture media, the observed CBF decreased, from 11−12 Hz to 6−8 Hz, as the concentration of cellulose increased from 0 to 1%. The CBF observed for each cellulosic derivative followed a similar trend at each concentration. These results are shown in Figure 4. This experiment suggested a correlation between CBF and polymer concentration. It is important to note CBF measurements could not be completed with PVCL−PVA−PEG because the solutions were opaque at 35 °C and distorted the view of the ciliated cells. 2736


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Incomplete recovery of acyclovir was observed following permeation experiments. Further drug recovery investigation was completed by assessing the drug detected via HPLC following incubation with the cellulosic derivative solutions. A known mass of acyclovir was dissolved in an aqueous solution and subsequently mixed with a desired concentration of cellulosic derivative. The solutions were incubated at 37 °C overnight, and the drug content was analyzed via HPLC. Figure 6 shows the percent of acyclovir that was lost following the incubation with the cellulosic derivatives. Little to no drug loss was observed when it was incubated with 0.1% MC and HPMC. Significant loss was observed when it was incubated with 0.1% sodium-CMC and cationic-HEC. To determine any absorption of acyclovir by the exposed tissue or interference by the cellulosic excipients during detection, a known mass of acyclovir was dissolved in an aqueous solution and subsequently mixed with a desired concentration of cellulosic derivative. One milliliter of these solutions was incubated with sections of nasal tissue of the same diameter as that used during the permeation study and incubated at 37 °C for 2 h, after which the drug content was analyzed via HPLC. All formulation types showed a significant decrease in the detected mass of acyclovir following incubation with nasal tissue ranging from 9 to 20%.

Figure 4. Effect of cellulosic polymer concentration on CBF at 35 °C.

Whether the reduction in CBF was temporary in the presence of the polymer solution or permanent due to physical or biochemical damage from the excipients was determined by evaluating the recovery of CBF after removing the polymer solution. As summarized in Table 2, all four cellulosic polymer derivatives showed only negligible differences in CBF before and after 20 min exposure to polymer solutions at various concentrations. The results strongly suggest that the cellulosic solutions did not cause any adverse effects on nasal epithelial cells for 20 min and confirmed that the reduction in CBF determined in the presence of polymer was indeed by an effect mediated by the polymer solution’s presence. Permeation Across Excised Porcine Mucosa. The permeation of a model drug, acyclovir, across excised porcine nasal tissue investigated using a Franz diffusion cell system showed a gradual increase during the first 15−30 min and then reached a constant rate for the rest of the measurement period, as shown in Figure 5. The permeability coefficients represent the efficiency of drug permeation through the given membrane and were determined in the presence of each polymer type. The use of cationic-HEC solution at 0.1% and a combination of cationic-HEC and 5% PVCL−PVA−PEG gave the highest permeability coefficient (Table 3), followed by acyclovir without any excipients present and then the remaining cellulosic derivatives (0.1% HPMC, 0.1% sodium-CMC, and 0.1% MC), which showed only marginal differences. The reported permeability coefficient for this drug is 6.21 ± 0.60 × 10−6 cm/s in porcine buccal epithelia.28 Our determined value in nasal epithelial tissues (8.9 ± 1.1 × 10−6 cm/s), without excipients present, is in a reasonable range compared to reported values when considering the similarity between the nasal epithelial structure and that of buccal epithelia.

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DISCUSSION METHOCEL derivatives (MC/HPMC in our study) have been known to possess a reversible thermogelling capability, but they often require significantly high concentrations to obtain a sufficiently low sol−gel transition temperature to be applicable in vivo, which is not suitable for spraying by nasal administration devices. Also, sodium-CMC possessed nearly no thermogelation capability on its own. Thermosensitive gelling as the temperature rises from room temperature to physiological temperature is a highly desirable property for intranasal drug delivery formulations because such an effect could contribute to a prolonged residence time of the formulation at the targeted nasal epithelium against the natural clearance mechanism resulting from the concerted motion of the ciliary beating.29,30 Our rheological data show that the reversible viscosity enhancement can be induced in the relevant temperature range by formulating with PVCL−PVA−PEG, which could be highly useful for nasal drug delivery applications. The reverse thermogelation of MC and HPMC is not fully understood, but it is hypothesized to occur as a result of hydrophobic interaction between molecules containing methoxyl substitutions.31,32 Briefly, in a solution state at lower temperatures, the polymer molecules are hydrated by a cagelike structure of water molecules with few polymer−polymer interactions. Upon heating, the hydrogen bonding of the water

Table 2. Ciliary Beat Frequency (CBF) in Hz of Porcine Nasal Mucosa Cells before and after Exposure to and Subsequent Removal of Cellulosic Derivative Solutions after exposure for 20 min cellulosic derivative concentration (w/w %) 1.00% 0.50% 0.25% 0.10% a

before exposure 10.8 10.4 10.8 10.4

(0.2)a (0.4) (0.3) (0.2)

MC 11.1 10.3 10.8 10.8

(0.4) (0.2) (0.3) (0.3)

HPMC 11.0 10.5 10.6 10.6

(0.3) (0.1) (0.4) (0.4)

sodium-CMC 11.0 10.3 10.6 10.6

(0.1) (0.1) (0.2) (0.4)

cationic-HEC 11.4 11.1 11.0 10.4

(0.2) (0.1) (0.4) (0.2)

Number in parentheses represents the standard error of the mean. 2737


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Figure 5. Comparison of the tissue permeability of acyclovir (2 mg/mL) in the presence and absence of cellulosic derivatives and PVCL−PVA− PEG: (a) 0.1% MC, HPMC, sodium-CMC, and cationic-HEC and (b) 0.1% cationic-HEC with and without 5% PVCL−PVA−PEG in ddH2O, pH 6.5.

Table 3. Summary of Tissue Permeability Results with Itemized Detailsa formulation type no polymer 0.1% MC 0.1% HPMC 0.1% sodiumCMC 0.1% cationicHEC 5% PVCL− PVA−PEG 0.1% cationicHEC + 5% PVCL−PVA− PEG

donor remaining

permeated

Peff

N

(%)

(%)

(cm/s × 10−6)

(replicates)

82.5 (4.1)b 56.6 (0.5) 51.8 (2.5) 65.9 (1.1)

5.9 4.2 4.8 4.8

79.5 (3.3)

(0.8) (0.6) (1.4) (1.1)

8.9 6.4 7.6 7.6

(1.1) (1.1) (2.3) (1.6)

11 3 3 3

7.6 (0.8)

11.6 (1.3)

12

87.0 (3.8)

5.5 (1.5)

8.4 (2.2)

4

87.1 (2.9)

9.9 (1.5)

14.5 (1.8)

5

a

Percent (%) donor remaining, % permeated, and permeability coefficient (cm/s) for each formulation. bNumber in parentheses represents the standard error of the mean. Figure 6. Percent of acyclovir lost during permeation across porcine nasal tissue to cellulosic derivatives and tissue.

molecules is weakened, causing partial dehydration and phase separation.33 The phase separation allows for hydrophobic polymer−polymer aggregation, forming junctions among polymer chains and resulting in three-dimensional networks (gelation).25 This phenomenon is reflected by a sharp rise in viscosity. Alternatively, the low-temperature viscosity increase induced by PVCL−PVA−PEG may be related to a clouding phenomenon of PVCL−PVA−PEG in aqueous solution.23,34 PVCL−PVA−PEG is a copolymer of three different polymer blocks: poly(vinyl caprolactam)−poly(vinyl acetate)−poly(ethylene glycol). Due to the amphiphilic nature of the copolymer composition, PVCL−PVA−PEG typically presents as a micelle structure in aqueous media. Upon heating, interaction between polymers is strengthened by dehydration, forming larger micelles that can increase light scattering. This clouding effect was observed in cellulose−PVCL−PVA−PEG mixture systems in our study as well. Although the elevation of viscosity was observed during clouding, this effect might not be

considered gelation in a conventional sense because the sol−gel transition (typically defined by G′/G″ = 1) did not occur. Although we did not perform experiments to fully understand the mechanism of interaction between cellulosic polymer and PVCL−PVA−PEG in the mixture, one possible hypothesis we propose is that the negatively charged cellulosic polymer, sodium-CMC, may induce a dipole interaction with the ester residue within the PVCL−PVA−PEG molecule, resulting in an enhanced maximal viscosity near physiologically relevant temperatures yet without lowering the onset temperature. In contrast, nonionic polymers (MC and HPMC), which enhance only the initial viscosity of the solution, may form pseudojunctions by entanglement through the amphiphilic PVCL−PVA−PEG molecule. This junction-like structure may be able to facilitate the hydrophobic interaction of PVCL− PVA−PEG, resulting in a shift in the gelation temperature. There was an initial concern in adding the polymers that they may substantially increase the viscosity at room temperature 2738


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effect of the presence of the cellulose polymers and not any loss in cell viability. The permeation of drug molecules across excised nasal mucosal tissue was evaluated in the presence and absence of 0.1% cellulosic derivatives. An antiviral model drug, acyclovir, was chosen for this study. Compared to a control, an acyclovir solution without any cellulosic derivative present, the counterparts with 0.1% MC, HPMC, and sodium-CMC exhibited slightly delayed permeation across the tissue membranes. This trend might be explained by the effect of solutions with slightly higher viscosities than that of pure aqueous media. While the other polymer types showed reduction in tissue permeation efficiency, cationic-HEC showed noticeably faster permeation than that of the no polymer control as well as the other cellulosic derivatives. This result may be explained by the cationic nature of this polymer. Cationic polymers, like chitosan, have been reported to possess a permeationenhancing property through various epithelial layers.36,37 The mechanism of action is still not fully elucidated, but the combined effect of mucoadhesion and a change in the gating of tight junctions is a generally accepted suggestion.20,37 The interior of the tight junction channels are known to be highly hydrated and negatively charged. The alteration of these properties, especially neutralization of charge by adsorption of cationic polymer, can lead the loosening of the pore.38 The permeation enhancement in the presence of cationic-HEC in our study may be explained by this same principle. Permeation of acyclovir across the excised tissues was further investigated by combining 0.1% cationic-HEC with 5% PVCL−PVA−PEG to observe any possible negative effects that the increased viscosity at 35 °C may have on the coefficient of permeability. The permeation of acyclovir from this formulation was consistent with the permeation from the 0.1% cationic-HEC formulation alone. From the permeation study, there was a significant amount of drug that was unaccounted for, as determined by analyzing the amount of drug permeated through the tissue and remaining in the donor chamber following the 2 h study as compared to the amount of drug in the initial formulation. The missing drug ranged from negligible to approximately 40% by mass. To account for this loss, the effect that the cellulosic derivatives and the presence of the tissue had on the quantification of acyclovir via HPLC was further investigated. No significant loss in drug was observed in the presence of MC and HPMC. A loss of approximately 5−8% of acyclovir by mass was observed in the presence of the charged sodium-CMC and cationic-HEC. This may be due to the known hydrogen-bond interactions that acyclovir can exhibit.39 The charged structures of sodium-CMC and cationic-HEC would provide the necessary moieties for these interactions to occur. No conclusive loss of drug was determined in the presence of cationic-HEC and PVCL−PVA− PEG combined. Significant loss of drug was observed when drug and cellulose derivative formulations were incubated with excised porcine nasal tissue. The most significant loss of drug to tissue was observed for the control solution, MC, and HPMC, which corresponds to the permeation trials with the highest percent of drug missing. An explanation of why more acyclovir is absorbed by tissue in these formulation types as compared to the sodium-CMC, cationic-HEC, and combined cationic-HEC and PVCL−PVA−PEG was not found. Despite further investigation, all of the drug was not recovered from some of the formulations tested. This can be attributed to variability in the nasal tissue thickness, which was not controlled, and can

and result in a formulation that was unsprayable. For this reason, we chose to utilize the cellulosic excipients at low concentrations. However, all of the viscosities observed are below the maximum viscosity previously found to produce acceptable spray patterns. Acceptability is dependent on the spray device, and design considerations can be optimized to produce the desired spray patterns.35 To assess bioadhesive polymer formulations, it is useful to determine a target value by which to indicate the success or failure of any given formulation. In the context of the thermogelation properties observed of the cellulosic derivatives and PVCL−PVA−PEG, a target viscosity could not be determined by in vitro testing alone. It is necessary to further explore these polymers in vivo to better evaluate the viscosity that would be required to obtain sufficient bioadhesion. Work to examine these polymers in vivo is ongoing. Ciliary beating is responsible for the mucociliary clearance defensive mechanism and is a major limiting factor for residence time of nasal drug formulations in the nasal cavity. Ideally, applying mucoadhesive polymers to extend the residence time should retard ciliary beating, but only transiently, without causing irreversible changes and toxicity to nasal mucosa. We evaluated the effect of the four cellulosic derivatives on ciliary beating frequency (CBF) in vitro. The reduction in CBF in the presence of cellulosic polymers was monitored by suspending brushed ciliated cells in polymer solutions. The potential toxicity was also evaluated by monitoring the recovery of CBF after exposure to polymer solution that was administrated on the tissue’s surface for 20 min. In the former method, CBF values decreased as the polymer concentrations increased. This trend seemed to suggest a correlation between CBF and viscosity. A direct correlation between viscosity and CBF could not be made according to our results. Upon further analysis, at 0.5 and 1% polymer concentrations, the viscosity of MC and HPMC is significantly higher than those of sodium-CMC and cationicHEC, in spite of similar ciliary beating values. An explanation for this phenomenon was not determined, but the effect could be due to the individual chemistry of the polymers. However, it is still known that viscosity plays a role in the CBF, and to this extent, we considered viscosity to be an important variable in this study.7 When considering a nasal spray drug product, there is a limit to the extent to which polymer viscosity can be raised in a nasal formulation to reduce CBF; otherwise, the initial viscosity of the solution may become too high to be sprayed from a nasal delivery device. Reversible viscosity increase by introducing PVCL−PVA-PEG to the cellulosic derivative is a potential solution to this issue. However, CBF testing using PVCL−PVA−PEG was not possible due to the clouding of PVCL−PVA−PEG at 35 °C, which prohibited viewing the ciliated cells. While the cellulosic derivatives had an observed effect on the CBF in vitro, work is ongoing to study the effects that these have on mucociliary clearance in vivo. In addition to the potential viscosity effect on CBF, the toxicity of cellulosic polymer solutions on nasal mucosa was assessed by monitoring changes in the CBF after a 20 min exposure (this duration was based on the reported turnover rate of mucous in the nasal cavity),34 as one important toxicity indicator. Compared to the control that was measured without exposure to any polymer solutions, all polymer solutions, up to 1% concentrations at the highest, showed only negligible effects on CBF recovery. This result strongly suggests that the retarded CBF in the presence of polymers was indeed caused by the 2739


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New developments and opportunities in oral mucosal drug delivery for local and systemic disease. Adv. Drug. Delivery Rev. 2012, 64, 16−28. (2) Ugwoke, M. I.; Agu, R. U.; Jorissen, M.; Augustijns, P.; Sciot, R.; Verbeke, N.; Kinget, R. Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa. Int. J. Pharm. 2000, 205, 43−51. (3) Patil, S. B.; Sawant, K. K. Chitosan microspheres as a delivery system for nasal insufflation. Colloids Surf., B 2011, 84, 384−389. (4) Andrews, G. P.; Laverty, T. P.; Jones, D. S. Mucoadhesive polymeric platforms for controlled drug delivery. Eur. J. Pharm. Biopharm. 2009, 71, 505−518. (5) Turker, S.; Onur, E.; Ozer, Y. Nasal route and drug delivery systems. Pharm. World Sci. 2004, 26, 137−142. (6) Zaki, N. M.; Awad, G. A.; Mortada, N. D.; Abd ElHady, S. S. Enhanced bioavailability of metoclopramide HCl by intranasal administration of a mucoadhesive in situ gel with modulated rheological and mucociliary transport properties. Eur. J. Pharm. Sci. 2007, 32, 296−307. (7) Ugwoke, M. I.; Agu, R. U.; Verbeke, N.; Kinget, R. Nasal mucoadhesive drug delivery: background, applications, trends and future perspectives. Adv. Drug Delivery Rev. 2005, 57, 1640−1665. (8) Behl, C. R.; Pimplaskar, H. K.; Sileno, A. P.; deMeireles, J.; Romeo, V. D. Effects of physicochemical properties and other factors on systemic nasal drug delivery. Adv. Drug Delivery Rev. 1998, 29, 89− 116. (9) Romeo, V. D.; deMeireles, J. C.; Gries, W. J.; Xia, W. J.; Sileno, A. P.; Pimplaskar, H. K.; Behl, C. R. Optimization of systemic nasal drug delivery with pharmaceutical excipients. Adv. Drug Delivery Rev. 1998, 29, 117−133. (10) Gizurarson, S. The relevance of nasal physiology to the design of drug absorption studies. Adv. Drug Delivery Rev. 1993, 11, 329−347. (11) Duan, X.; Mao, S. New strategies to improve the intranasal absorption of insulin. Drug Discovery Today 2010, 15, 416−427. (12) Garg, N. K.; Mangal, S.; Khambete, H.; Tyagi, R. K. Mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymers. Recent Pat. Drug Delivery Formulation 2010, 4, 114−128. (13) Nazar, H.; Fatouros, D. G.; van der Merwe, S. M.; Bouropoulos, N.; Avgouropoulos, G.; Tsibouklis, J.; Roldo, M. Thermosensitive hydrogels for nasal drug delivery: the formulation and characterisation of systems based on N-trimethyl chitosan chloride. Eur. J. Pharm. Biopharm. 2011, 77, 225−232. (14) Lehr, C. M.; Bouwstra, J. A.; Schacht, E. H.; Junginger, H. E. In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. Int. J. Pharm. 1992, 78, 43−48. (15) Park, J. S.; Oh, Y. K.; Yoon, H.; Kim, J. M.; Kim, C. K. In situ gelling and mucoadhesive polymer vehicles for controlled intranasal delivery of plasmid DNA. J. Biomed. Mater. Res. 2002, 59, 144−151. (16) Mayo, L.; Quaglia, F.; Borzacchiello, A.; Ambrosio, L.; La Rotonda, M. I. A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: rheological, mucoadhesive and in vitro release properties. Eur. J. Pharm. Biopharm. 2008, 70, 199−206. (17) Bernkop-Schnurnch, A.; Hornof, M.; Zoidl, T. Thiolated polymers-thiomers: synthesis and in vitro evaluation of chitosan-2iminothiolane conjugates. Int. J. Pharm. 2003, 260, 229−237. (18) Hornof, M. D.; Kast, C. E.; Bernkop-Schnurch, A. In vitro evaluation of the viscoelastic properties of chitosan−thioglycolic acid conjugates. Eur. J. Pharm. Biopharm. 2003, 55, 185−190. (19) Bernkop-Schnurch, A.; Scholler, S.; Biebel, R. G. Development of controlled drug release systems based on thiolated polymers. J. Controlled Release 2000, 66, 39−48. (20) Davis, S. S.; Illum, L. Absorption enhancers for nasal drug delivery. Clin. Pharmacokinet. 2003, 42, 1107−1128. (21) Ugwoke, M. I.; Kaufmann, G.; Verbeke, N.; Kinget, R. Intranasal bioavailability of apomorphine from carboxymethylcellulose-based drug delivery systems. Int. J. Pharm. 2000, 202, 125−131. (22) Mazoniene, E.; Jocevivute, S.; Kazlauske, J.; Niemeyer, B.; Liesiene, J. Interation of cellulose-based cationic polyelectrolytes with mucin. Colloids Surf., B 2011, 83, 160−164.

affect the amount of drug absorbed. Additionally, accurate quantification of drug in a gel is associated with increased experimental error.

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CONCLUSIONS Four types of cellulosic polymer derivatives, MC, HPMC, sodium-CMC, and cationic-HEC, have been examined based on various categories that are relevant parameters for nasal drug delivery applications. Inclusion of PVCL−PVA−PEG with all four cellulose polymers resulted in a reversible enhancement in viscosity at the physiological temperature of the nasal cavity. PVCL−PVA−PEG combined with sodium-CMC suggested a possible synergistic effect when the viscosity enhancement was observed. None of the cellulosic derivatives caused an irreversible change in the CBF. Cationic-HEC was observed to enhance the transport of drug molecules across the nasal epithelium. PVCL−PVA−PEG has also been shown by these data to be an excipient of interest, in combination with these cellulosic derivatives, for enhancing the studied performance characteristics of nasal drug formulations. These findings clearly demonstrate certain potential advantages of utilizing these cellulosic polymers in nasal drug delivery applications. However, none of these polymers stand out on their own for their ability to provide more than one merit. In the future, we hope to use this understanding alongside these techniques to develop more tailored polymer nasal formulations for important drug molecules.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Mean TEER pattern, as seen from the continuous measurements on individually mounted tissue before and after exposure to acyclovir formulations. Table S1: Mean TEER values ± SEM of the last 10 minutes of the final stabilization period for all excised porcine nasal tissues and corresponding ranges from the literature. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00264.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ⊥

K.H., G.K., and K.-G.H.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by Dow Chemical Company. The authors are grateful to the ASOR and ECMO laboratories at the University of Michigan Health System for the supply of porcine nasal mucosa as well as Milligan’s Northwest Market (Jackson, MI). The authors also wish to thank J. Maxwell Mazzara for assistance with porcine nasal tissue harvesting. METHOCEL, WALOCEL, and UCARE are trademarks of The Dow Chemical Company (Dow) or an affiliated company of Dow.

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REFERENCES

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