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Bioconjugate Chem. 2008, 19, 2394–2400
Investigation of Insulin Loaded Self-Assembled Microtubules for Drug Release Marsiyana M. Henricus, Karen T. Johnson, and Ipsita A. Banerjee* Department of Chemistry, Fordham University, 441 East Fordham Road, Bronx, New York 10458. Received June 25, 2008; Revised Manuscript Received September 22, 2008
Self-assembled microtubules were used to entrap insulin for the preparation of new drug delivery devices. The interactions of insulin with the microtubules were probed by circular dichroism, zeta potential analysis, as well as FTIR spectroscopy. The morphologies of the insulin-loaded tubules were examined by AFM and TEM. We found that insulin loading was both pH- as well as concentration-dependent. The circular dichroism analysis indicated that, at pH range 6-7, the conformation change in the presence of the microtubules was minimal and hence would be the most appropriate conditions for insulin loading. The entrapment efficiency and release of insulin was found to be pH-dependent. Further, the controlled drug release studies indicated that, under acidic conditions, insulin release was extremely slow, and it is likely that the insulin is protected inside the microtubules. Thus, the microtubules may potentially protect the insulin from aggregation and release at lower pH (gastric pH) in ViVo. However, at pH 6.5 (closer to intestinal pH) a sustained release was observed. Such new materials may inhibit the aggregation of peptides under suitable conditions and potentially be used for drug delivery, in particular, for other peptide-based drugs.
INTRODUCTION Over the past several years, attention has been focused on research related to the development of nanomaterials for numerous applications such as biosensors, optoelectronics, catalysts, and drug delivery systems (1-4). In addition to their small size, nanomaterials can be altered to facilitate attachments to specific components, in order to improve biocompatibility and allow for more accurate intracellular and intercellular targeting (5-7). Nanoparticles consisting of polymers, ceramics, metals, as well as biological materials have been constructed to form various types of structures that offer unique characteristics to aid drug delivery (8-11). Many of the polymeric nanoparticles that have been developed are biodegradable, preventing the accumulation of the nanoparticles in the body in addition to ensuring the constant drug release over a period of time. Soft delivery systems such as surfactant micelles, liposomes engineered from phospholipids, and vegetable oil based materials have also been used to carry various types of biological materials and lipophilic drugs. In lieu of this, several lipid formulations have been developed to transport protein drugs and allow for their sustained release (12-22). A rapidly growing drug delivery approach is the use of microtubules and nanotubes because of their large relative internal volumes, which can be functionalized on the internal and the external surface. Self-assembled lipid microtubes and carbon nanotubes (CNTs) have been utilized as drug delivery agents (23-27). Nanotubes and microtubules self-assembled from peptide bolaamphiphiles exhibit several properties that make them promising biomaterials, including facile selfassembly in aqueous solutions, biocompatibility, and ability to be functionalized (28, 29). For example, self-assembled peptide nanotubes have been used to immobilize enzymes such as lipase, which upon immobilization showed improved stability (30). * Author to whom all correspondence should be addressed. I. A. Banerjee. Phone: 718-817-4445. Fax: 718-817-4432. E-mail: banerjee@ fordham.edu.
In this work, the peptide drug, insulin, was entrapped within self-assembled peptide microtubules. Insulin is an important hormone for the regulation of blood glucose levels and is implicated in diseases such as Diabetes type I and type II (31). Aggregation of insulin and consequent precipitation has been a major barrier to the development of long-term delivery methods for insulin (32, 33). Many methods have been used to protect insulin and other peptide drugs from physical inactivation, such as the addition of stability-enhancing additives, surfactants, and phenols (34, 35). Nanomaterials such as poly(alkyl cyanoacrylate), PLGA, poly(vinyl alcohol) gel spheres, hydroxypropyl methylcellulose phthalate microspheres, Eudragit L100 microspheres, and poly(γ-glutamic acid) nanoparticles shelled with chitosan have been functionalized with insulin for purposes of drug delivery (36-40). It is well-known that dicarboxylic amino acids have been successfully used to prevent agitation-induced aggregation in insulin (41). However, the rich potential of selfassembled tubular assemblies formed from synthetic amino acid bolaamphiphiles (two amino acid head groups connected by a hydrophobic tail group) for diminution of aggregation, entrapment of insulin, and preparation of a potential drug delivery device is yet to be explored. Here, we have examined highly biocompatible microtubules self-assembled from bis(N-R-amidotyrosyl-tyrosyl-tyrosine)-1,5-pentane dicarboxylate for controlled drug release of insulin. The main objective of the present study was to examine the interactions of insulin with the microtubule, determine entrapment efficiency, and study their release behavior. Such materials may potentially be useful for the encapsulation of other peptides and drugs and promote longer circulation times in ViVo.
EXPERIMENTAL SECTION Materials. 1-Hydroxy-benzotraizole hydrate, Tyr-Tyr-Tyrmethyl ester, DMF, EDAC (dimethylaminopropyl-N-ethylcarbodiimide hydrochloride), chloroform, methanol, TEA (triethyl amine), NaHCO3, NaOH, HCl, and insulin from Porcine pancreas (g27 USP units/mg) were purchased from Sigma Aldrich. Phosphate buffer solutions of various pH values were
10.1021/bc800254n CCC: $40.75 2008 American Chemical Society Published on Web 11/06/2008
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Figure 1. (a) TEM image of a self-assembled microtubule. (b) TEM image of partially loaded insulin microtubule at 0.2 mg/mL concentration of insulin. (c) Uniformly loaded insulin microtubule at 0.5 mg/mL concentration of insulin.
prepared, and the pH was adjusted by adding either HCl or NaOH as required; Brillant Blue G-250, 95% ethanol, and phosphoric acid were purchased from Fisher Scientific. All chemicals were used as received. Methods. Growth and Self Assembly of Microtubules. The bolaamphiphile was synthesized and self-assembled according to previously established methods (28, 29, 42). Briefly, the bis(N-R-amido-tyr-tyr-tyrosine)-1,5-pentane dicarboxylate (8 mM) was self-assembled into microtubules at pH 5 over a period of two weeks. The assembled tubules were then sonicated, washed, and centrifuged twice at 4000 rpm for 3 h before incorporation of insulin. Preparation of Insulin-Loaded Microtubules. Various concentrations of insulin in the range of 0.1 mg/mL to 2 mg/ mL were incubated with the microtubules for a period of 72 h at 4 °C while mildly agitating the samples. The samples were then centrifuged and washed twice with nanopure water to remove any un-entrapped insulin. In order to determine the optimum pH for entrapment of insulin, the experiments were carried out in the pH range 4-10. Measurement of Entrapment Efficiency. The amount of insulin loaded into the peptide microtubules was determined by measuring the difference between the total amount of insulin added into the peptide microtubules and the amount of unloaded insulin present in the supernatant after the loading process. In general, a concentration of 0.5 mg/mL of insulin was used in order to carry out the entrapment efficiency studies. The studies were carried out in varying pH, to determine the effect of pH on the loading capacity. The loaded microtubules (500 µL) were centrifuged at 4500 rpm at 4 °C for 5 h, and then the amount of free insulin in the clear supernatant was measured by the Bradford method (43). The entrapment efficiency was calculated using the following equation: (X1 - X2/X1) × 100%, where X1 is the concentration of the amount of insulin and X2 is the concentration of the unloaded insulin. Therefore, entrapment efficiency of insulin in microtubules )
total amount of insulin used - free insulin in supernatant × 100 total amount of insulin used (1)
Drug Release Studies of Insulin. The insulin-loaded microtubules (formed at pH 6) were washed and centrifuged thoroughly before conducting the release studies. In order to determine the pH effect on the release studies of insulin from the microtubules, we investigated the release of insulin at varying pH. Insulin-loaded microtubules (500 µL) were added to 1.5 mL of respective buffer solutions (pH 4-10) and the amount of insulin released was measured via the Bradford method. Measurements were carried out for a period of 5 h at 37 °C. All measurements were carried out in triplicate.
Characterization. Circular Dichorism (CD) Spectroscopy. CD measurements were carried out using a JASCO J-720 spectropolarimeter. Samples were scanned at least five times at the rate of 200 nm/min with a 0.5 nm step, 1 nm bandwidth, and then averaged. The path length of the quartz cell was 2 mm. In the experiments, a blank run made with buffer alone was subtracted from the experimental spectra for correction. The 190-250 nm spectra were used for analysis. All the spectra were smoothed and converted to the mean residue ellipticity [θ] in deg*cm2/dmol. FTIR Spectroscopy. In order to confirm the incorporation of insulin onto the microtubules, FTIR analyses were performed using Matteson Infinity IR equipped with DIGILAB, ExcaliBuv HE Series FTS 3100 software. The samples were dried at room temperature and mixed with KBr to make pellets and then analyzed. All spectra were taken at 4 cm-1 resolution with 50 scans taken for averaging. Sample measurements were carried between 400 and 4000 cm-1. Absorbance Spectroscopy. To determine the protein concentration using the Bradford method, absorbance measurements were carried out at λmax of 595 nm using a Varian Cary 3 UV-visible spectrophotometer. Samples were diluted with appropriate amounts of buffer or nanopure water. Zeta Potential Analysis. A NICOMP 380 ZLS zeta potential/ particle sizer system (Santa Barbara, CA) was used to determine the zeta potential of the samples. Measurements of ζ-potential of the samples were carried out at 25 °C and at varying pH. The concentrations of the suspensions were adjusted within the operational limits of the instrument, and the suspension pH was adjusted by the addition of standard buffer solutions. The reported ζ-potential values are the average of at least five measurements (spread of values ( 5% of the reported mean values). Atomic Force Microscopy. Atomic force microscopy was carried out using a Quesant Universal SPM Instrument by the tapping mode in air using a silicon nitride cantilever. Transmission Electron Microscopy (TEM). The morphologies of the samples were analyzed by TEM (JEOL 120 EX) operated at 100 kV. Samples were washed twice and air-dried on carboncoated copper grids before analysis.
RESULTS AND DISCUSSION The microtubules were self-assembled in aqueous solution at pH 5. Both hydrogen bonding interactions between the amide and carboxyl groups as well as the hydrophobic interactions allowed for the facile self-assembly of the bolaamphiphile. The microtubules formed via self-assembly at room temperature within a period of two weeks. Details of the self-assembly of the microtubules are described elsewhere (29). A TEM image of a self-assembled microtubule before entrapment of insulin
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Figure 2. (a) TEM image of insulin-coated microtubules on the outside wall, at 1 mg/mL concentration of insulin. (b) AFM amplitude image of insulin-coated microtubules. Inset shows a single coated microtubule in the presence of a high concentration of insulin (1 mg/mL).
is shown in Figure 1a. The morphologies of the tubules after incorporation of insulin were examined by transmission electron microscopy and atomic force microscopy. On an average, we used tubules with an inner diameter of 500 nm to entrap the insulin. In order to determine optimum conditions for incorporation of insulin, it was incubated with the microtubules at varying pH for different periods of time. The concentration of insulin was also varied. By and large, we observed that, after 72 h, insulin was entrapped within the microtubules. The results obtained indicate that the incorporation of insulin was found to be not only pH-dependent but also concentration-dependent. At concentrations between 0.5 mg/mL and 0.6 mg/mL, at a pH range between 4 and 7, after incubation for 72 h, insulin was entrapped inside the microtubules. Figure 1a shows the TEM image of a microtubule before incubation with insulin. Figure 1b shows the TEM image of a microtubule after incubation for 72 h at pH 6 when the sample was incubated with 0.2 mg/mL insulin, while Figure 1c shows the TEM image of a microtubule where in the insulin is uniformly encapsulated after 72 h at pH 6 in the presence of 0.5 mg/mL insulin. As shown in figures 1b and 1c, the insulin appeared as darker areas in the microtubules (44). We also observed that, at higher pH, insulin tended to form aggregates and was not incorporated uniformly within the microtubules even at low concentrations (data not shown). At higher concentrations (between 0.8 mg/mL and 2 mg/mL), the insulin bound to the outside surface of the microtubules as well (Figure 2). The TEM image of insulin-microtubule (Figure 2a) shows the incorporation of insulin within the microtubules as well as formation of aggregates on the outside walls of the tubules. Those studies were also carried out after 72 h. The concentration of insulin used therein was 1 mg/mL. Figure 2b shows the AFM image of insulin-coated microtubules at pH 6, when the samples were incubated with 1 mg/mL of insulin. The inset shows a single microtubule, whose outside walls are coated with insulin. Thus, at lower concentrations, it appears that the inside loading mechanism of insulin may be similar to incorporation of nanoparticles via capillary effect as observed in previous work where the trends were similar (45, 46), while at higher concentrations, they tend to adsorb onto the outside walls of the microtubules as well. Although the exact mechanism of entrapment of insulin is not clear yet, it is likely that, as the microtubules are exposed to the insulin solution, it diffuses inside due to a concentration gradient as well as due to capillary action. Hydrophobic interactions between the tyrosines of the
Figure 3. Comparison of CD spectra of insulin and insulin-loaded microtubules at varying pH.
bolaamphiphiles and the hydrophobic residues of insulin may also be involved. In addition, electrostatic interactions and hydrogen bonding also play a critical role. Circular Dichroism. The conformational changes of insulin upon association with microtubules was evaluated using CD spectroscopy. Figure 3 indicates the CD spectra obtained in the presence and absence of microtubules under different pH conditions. At pH 4, the CD spectrum of insulin shows negative ellipticity at 208 and 222 nm as well as a positive band with a maximum at 192 nm. These peaks are indicative of typical R-helical structure (47). After incubation with the microtubules, however, there is a decrease in the negative ellipticity at 222 nm as well as 208 nm. This decrease in intensity upon binding is indicative that there is a reduction in the R-helical content; further, this indicates that interaction of insulin with microtubules at pH 4 may result in partial unfolding of the protein structure. Increasing the pH to 6.8 showed a reduction in the R-helical content of insulin by itself compared to that observed at pH 4; however, when bound to the microtubules, we did not observe significant changes in the secondary structure, except that there is a shift in the negative ellipticity band from 208 to 206 nm and a reduction in the intensity of the positive maxium
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Table 1. Zeta Potential Analysis Comparison of Insulin Loaded Microtubules pH pH pH pH pH pH
4 5 6 7 9
Zeta Potential of Microtubules (mV)
Zeta Potential of Insulin-Loaded Microtubules (mV)
5.96 5.51 1.24 0.18 -1.81
6.22 6.72 2.19 2.17 -1.69
observed at 194 nm. At pH 9, however, upon incorporation of the microtubules, the protein seems to undergo unfolding, forming a random coil like structure, with a negative ellipticity band at 194 nm, and its structure is significantly different from that observed for insulin by itself in solution at pH 9. Also, at pH 9, the R helix content of insulin appears to be significantly less than that observed at lower pH and the CD spectrum becomes one that is more typical of that observed for β-sheet structures, characterized by a minimum in the ellipticity at 216 nm and a maxima at 196 nm. This trend is similar to previous observations of insulin CD studies (48). These studies indicate that the optimum pH for entrapping insulin within the microtubules would be around pH 6-7, as the conformation change was relatively minimal under those conditions. Zeta Potential Analysis. The pH dependence on the ζ-potential of the tubules before and after entrapment of insulin is shown in Table 1. We observed that the ζ-potential of insulinentrapped tubules was slightly higher compared to those of the tubules by themselves. Although changes in the ζ-potential are evident, no drastic changes were observed, which indicate that insulin was encapsulated within the tubules and not adsorbed on the outside as well. This effect is similar to those observed in the case of polymeric nanocapsules, wherein ζ-potential changes were negligible upon encapsulation (49). We observed that under basic conditions the ζ-potential of the insulin-tubules remained negative. Since the isoelectric point of insulin is 5.5, above its pI, insulin becomes negatively charged, resulting in favorable electrostatic interactions between microtubule and insulin and increased loading up to pH 7. At basic pH, the negatively charged insulin would have less attraction toward the negatively charged microtubules, resulting in significantly less incorporation of insulin due to the presence of deprotonated residues of insulin as well as the COO- groups of the microtubules. Infrared Spectroscopy. In order to confirm the incorporation of insulin, the samples were also analyzed by FTIR spectroscopy. Figure 4 shows the comparison of IR spectra of insulin and insulin-loaded tubules obtained at pH 6, when 0.5 mg/mL concentration of insulin was used. In the insulin-loaded tubules, peaks at 1293 cm-1 and 1269 cm-1 due to the CsO stretching vibrations are slightly blue-shifted compared to those observed for insulin at 1286 cm-1 and 1261 cm-1 due to CsOsH hydrogen bonding interactions (50, 51) between the microtubules and insulin. Peaks at 1487 cm-1 due to CdC aromatic stretching vibration seen in insulin are shifted to 1485 cm-1 in the insulinloaded tubules. This peak is further shifted from the weak CdC aromatic stretching peak observed for the microtubules by themselves at 1492 cm-1 due to the presence of the tyrosine moieties in the microtubules. Peaks at 1592 cm-1 and 1569 cm-1 due to the amide NsH (1°-amide) II band and NsH (2°amide) II band observed in the case of insulin-loaded microtubules are also blue-shifted to a small extent compared to those of insulin at 1590 cm-1 and 1567 cm-1, respectively. Characteristic asymmetric stretching due to COO- occurring in the region 1410-1460 cm-1 and a small peak at 1562 cm-1 are seen due to the amide II NsH bend of the microtubules. In the case of insulin alone (Figure 4a), an intense peak is observed at 1659 cm-1, which is attributed to the amide I stretching
Figure 4. Comparison of FTIR spectra of insulin-loaded microtubules, insulin, and microtubules.
vibration and typical for R-helix conformation (52). This peak is also seen in the case of insulin-loaded tubules at 1652 cm-1, though less intense due to interactions with the tubules indicating that the conformation of insulin is relatively similar in the microtubules. Overall, the broad peak centered around 1620 cm-1 for the insulin-loaded microtubules is mostly due to the combined contributions of both insulin and the microtubules, particularly the amide I CdO stretches (free and H-bound) at 1654 cm-1and 1639 cm-1, as well as the carboxylate peak at 1721 cm-1 of the microtubules. No distinct peak at 1721 cm-1 is observed for the insulin-loaded microtubules, indicating the involvement of COOHsHOOC interactions with insulin. It is to be noted that similar shifts were observed in the case of pH 4 samples as well (data not shown). These results further confirm that the insulin was loaded in the microtubules. Entrapment Efficiency and in Vitro Release Behavior of Insulin Microtubules. The drug carrying capacity or entrapment efficiency of delivery systems is vital when determining the efficacy of a particular system. The entrapment efficiency of insulin within the microtubules was examined at various pH. The concentration of insulin was kept constant at 0.5 mg/mL, and the amounts of insulin encapsulated at various pH were calculated. As expected, the entrapment efficiency was found to be pH-dependent. This effect is shown in Figure 5. It was observed that the entrapment efficiency was highest in the pH 6-7 range At pH 6.8, the entrapment efficiency was found to be 80.5%, which was calculated to be 0.382 mg/mL. Subsequently, the entrapment efficiency became less at basic pH. It is most likely that there is charge repulsion between the deprotonated carboxylate groups of the microtubules and deprotonated amino acids of insulin at basic pH, leading to lesser
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Figure 5. Effect of pH on the entrapment efficiency of insulin in the microtubules.
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characterized by an initial rapid release during the first hour (for pH 6.5 and 7.4) followed by a relatively sustained release. It appears that insulin release takes place via disassociation of insulin from the microtubules. At low pH, the dissociation of insulin is relatively less as the microtubules are highly protonated, and the strong hydrogen bonding interactions may allow for less insulin to be released. As the pH is increased, the release of insulin increases. Although in ViVo conditions may vary, the microtubules appear to have higher insulin retention capacity at low pH, and thus, it is likely that under acidic conditions (for example, in gastric environment) the insulin may be protected inside the microtubules and may be more likely to release at intestinal pH (pH 6-7) where the absorption and action of the drug are necessary. Such materials may be applicable for insulin drug delivery systems. In order to further improve the conformational stability of the insulin within the microtubules while optimizing their delivery, future work will involve spray freeze-drying the insulin-loaded microtubules. The rapid freezing of the insulin-loaded microtubules may prevent phase separation of the drug, thwart crystalline growth in frozen water, and result in a more stable insulin delivery system. Further in ViVo studies are ongoing and will be published separately.
CONCLUSIONS In this work, we have utilized self-assembled microtubules formed from the peptide bolaamphiphile bis(N-R-amidotryosyl-tyrsol-tyrosine)-1,5-pentane dicarboxylate for entrapment of insulin. The interactions of insulin with the selfassembled microtubules were probed by circular dichroism, zeta potential analysis, as well as FTIR spectroscopy. The morphologies of the insulin-loaded tubules were examined by AFM and TEM. We found that insulin loading was both pH as well as concentration-dependent. The circular dichroism analysis indicated that, at pH 6-7, the conformation change upon interaction with microtubules was relatively minimal and hence would be the most appropriate conditions for insulin loading. The entrapment efficiency and release of insulin was found to be pH-dependent. Further, we also carried out controlled drug release studies, which indicated that, under acidic conditions, the insulin release was extremely slow, and it is likely that the insulin is protected inside the microtubules. However, at pH 6.5-7.4, a sustained release pattern was observed. Figure 6. Effect of pH on the in Vitro release of insulin from microtubules.
entrapment efficiency. Further, at higher pH, insulin tends to aggregate, making it more difficult for incorporation into the microtubules. A key benefit of this process is that insulin loading can be accomplished without the use of organic solvents or the addition of external additives. The release of insulin was studied over a period of 5 h at 37 °C at varying pH. The in Vitro release profiles of insulin from the microtubules at different pH are shown in Figure 6. In general, some of the drawbacks of peptide drugs include degradation in the stomach and inactivation by proteolytic enzymes in the luminal cavity. In order to mimic the conditions in the stomach, we studied the insulin release patterns at low pH (pH 1-3). We observed that, at low pH, the release was significantly slower, whereas a sustained release pattern was obtained at a pH of 6.5 (closer to the conditions in the duodenum). At pH 7.4 (closer to conditions at ileum), we observed a similar trend as that observed at pH 6.5, although the initial release was relatively faster. Overall, the release pattern of insulin was found to be pH-dependent and was
ACKNOWLEDGMENT The authors thank Dr. Areti Tsiola at the Queens College Core Facilities Center for Biomolecular imaging for the use of the transmission electron microscope and Ed Nieves at the Albert Einstein School of Medicine for the use of the Circular Dichroism spectropolarimeter. I.B. thanks the Office of Research at Fordham University for the Faculty Research Grant support.
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