Carrier-Based Drug Delivery - American Chemical Society

Chapter 3

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Proniosome-Derived Niosomes for the Delivery of Poorly Soluble Drugs 1,3

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David G. Rhodes and Almira Blazek-Welsh 1

Lipophile Consulting, 93 Timber Drive, Storrs, CT 06268 University of Connecticut, Storrs, CT 06269 Current address: PowderJect Vaccines, Inc., 585 Science Drive, Madison, WI 53711 (email: [email protected]) 2

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A novel approach to delivery of hydrolyzable, poorly soluble drugs is described. The method is based on a liposome pro -duction method using "proniosomes." These proniosomes consist of maltodextrin powder coated with surfactant or a surfactant/drug mixture to yield a dry powder. Upon addition of hot water and brief agitation, the maltodextrin dissolves and the surfactant forms a suspension of multilamellar vesicles (niosomes) containing the poorly soluble drug. The niosomes slowly release drug into solution. The proniosome powder can also be mixed with hydrogel powder. Adding hot water to the mixed powders allows formation of a hydrogel in which niosomes spontaneously form. The niosome-containing hydro -gel can be formulated as a gel that will degrade and release intact niosomes or as a stable gel, which slowly releases the drug from niosomes that remain in side the gel matrix.

© 2004 American Chemical Society

In Carrier-Based Drug Delivery; Svenson, Sönke; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction Because many pharmaceutical actives are poorly soluble, formulating them has been an ongoing problem for pharmaceutical scientists. Poorly soluble drugs can be formulated as oil-filled capsules or emulsions, for example, or can be chemically modified by conversion to salts or prodrugs. In addition to solubility concerns, some drugs are susceptible to hydrolysis and must be protected from water in their formulation and packaging. One approach to formulating poorly soluble drugs is to use liposomes. (1) The number of approved lipid-based drug formulations has been slowly increasing, but there remain certain stability issues. Liposomes are large particles and tend to precipitate out of suspension upon prolonged storage. Depending upon the chemical composition and physical properties of the liposomes, they can also aggregate, thus exacerbating the precipitation problem. In addition to these physical stability issues, liposome suspensions can suffer from chemical instability due to the presence of water. Hydrolysis of the surfactant can result in destabilization of the liposomes and/or alteration of the release properties of the suspension. Hydrolysis of the active can result in inactivation of the product and thus a shortened shelf life. Liposomes can be produced by any of a number of methods and the properties of the resulting liposomes will be determined by the choice of method. The most common approach involved drying surfactant (lipid) from organic solvent into a thin film (Figure I). (2) The drying is normally performed in a round bottom flask on a rotary evaporator so the surface area available for the film is typically on the order of 10 cm and the films are tens of jim thick. Once the film has been thoroughly dried, it can be rehydrated with aqueous medium at a temperature above the phase transition temperature (T ). The lipid film takes up water and begins to swell. In the absence of agitation, very large unilamellar vesicles can form as large "blebs'* before breaking off into solution. (3) With agitation, shearing forces result in formation of multilamellar vesicles. The time of agitation required to recover the lipids from the surface of the flask can be significant and the size distribution of the multilamellar vesicles can be broad and poorly reproducible. Further processing of the multilamellar vesicles to form unilamellar vesicles of desired size is possible. Liposomes can be used to formulate soluble drugs by entrapping the drugs in the aqueous solvent between lipid bilayers or the aqueous volume inside unilamellar vesicles. (I) Because the aqueous volume per lipid is smaller in multilamellar vesicles than in unilamellar vesicles, unilamellar vesicle are often preferred for drugs with good solubility. In liposomal formulations of poorly soluble drugs, the drug can be partitioned into the bilayer interior or at the bilayer interfacial region between the aqueous exterior and the hydrophobic core. For uniformly non-polar molecules, x-ray and neutron diffraction experi ments have demonstrated that the molecules partition to the bilayer center, while 2

2

m



42 amphiphilic drugs stay near the bilayer surface. (4) The association of poorly soluble drugs with liposomes is thus a partitioning process rather than an entrapment per se, but calculations of "entrapment efficiency" do not normally make this distinction. Vesicles can also be formed using nonionic surfactants such as Span 60, but these are more stable if some ionic surfactant and/or cholesterol is added. Nonionic surfactant liposomes, referred to as "niosomes", have been widely studied and are used in commercial preparations. Niosomes are normally multi lamellar and are formed using a methodology similar to that used for liposomes. (5) The advantages usually cited for using nonionic surfactants include cost of materials, availability, and chemical stability. The dosage forms described in this chapter were designed to overcome several significant issues encountered in formulating drugs in liposomes. In particular, (a) the formulation allows liposomes or niosomes to be produced "as needed," hydrating the suspension only when ready to use, (b) significantly reduces the agitation time required to form the liposomes, (c) improves the quality and consistency of the resulting liposomes or niosomes, (d) allows for flexibility in the dosing, and (e) provides a platform for new dosage forms based on the proniosome system. The system is suitable for use with nonionic surfacetants or phospholipids, but the data described here are for nonionic surfactants, and the text refers to "niosomes".

Figure I. Hydration of a dry lipidfilmabove the phase transition temperature T and subsequent agitation yields multilamellar vesicles, which can then further processed. m


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Results and Discussion Proniosome-Based Niosomes Most proniosome-based niosomes are mixtures of Span 60 (mixture of sorbitan stearate and palmitate), cholesterol, and dicetyl phosphate. These nonionic surfactant-based systems are less expensive than phospholipid-based systems and are generally less susceptible to degradation of the surfactant. Nevertheless, some of the same problems persist. If a suspension is made ahead of time, there is a risk of physical or chemical instability. If the formulation is hydrated "as needed," problems of lengthy rehydration time, inconsistent reconstitution, and lack of flexibility in the dose are still present. Hu and Rhodes demonstrated that niosomes could be produced from a dry, sorbitol-based formulation. (6) In this system, a sorbitol powder was sprayed with surfactant in chloroform and dried. Because the sorbitol was soluble in chloroform, however, the process required repeated spray/dry cycles so that the powder morphology was not significantly altered. Compared to the conventional approach, in which surfactant is dried onto the inner surface of a glass container, this approach provided a much higher surface area, and thus a much thinner coating. The niosomes were reconstituted by adding aqueous buffer at 60°C and agitating the mixture (Figure 2). Compared to conventional niosomes, the proniosomederived niosomes were slightly smaller and more uniform in size (Figure 3). For the model systems tested, hydrophilic drugs were not entrapped efficiently, but hydrophobic drugs could easily be included in these multilamellar niosomes. Release of the entrapped drug was slightly slower than observed for conven tional niosomes. powder particle

spray with surfactant

Niosomes

very thin coating

Proniosome dissolve in hot water

(in dissolved carrier)

0

o

°

surfactant hydrates

I

\

\

powder * dissolves

Figure 2. Proniosomes are a dry formulation made by coating a water-solu "carrier" powder with a thinfilmof surfactant. Rehydration of the dry pro some preparation by addition of a hot aqueous phase and brief agitation res in the formation of multilamellar vesicles in a dilute solution of carrier.



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Figure 3. Niosomes produced using conventional methods (a) and derive proniosomes (b). Aside from the difficulty of repeated spray/dry cycles, one of the most signifycant problems with this system was the presence of residual carrier. The presence of large amounts of dissolved sorbitol was observed to affect the entrapment of drug. A large number of alternative substrates was explored (see acknowledgements), including soluble and insoluble materials. Eventually, a material was identified that was soluble in water but not in chloroform, the solvent used to dissolve the surfactants. (7) Maltodextrin, obtained from GPC, is available as solid particles (Maltrin QD M500) with a bulk density 0.34 g/cm or hollow particles (Maltrin M700) with a bulk density of 0.13 g/cm (Figure 4). 3

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Figure 4. Electron micrographs of maltodextrin powder. The solid powd (M500, left) has a rough texture, while the hollow-blown powder (M700 is a thin, smooth shell. 2

The surface area of the hollow product was estimated to be 8000 cm /g, approximately 20 times greater than that of M500. Because the powders were insoluble in chloroform, the preparation was very simple. The desired pro portions of surfactant and poorly soluble drug in a chloroform solvent were added to maltodextrin powder in a rotary evaporator flask. The mixture was subjected to agitation and vacuum to remove the solvent, resulting in a dry powder. The powder, surfactant coated maltodextrin, was then kept under vacuum overnight to assure that the solvent was completely removed. Niosomes were made by adding hot (60°C) water to a vial of proniosome powder and shaking the vial for at least one minute. It was important to keep the temperature


45 of the aqueous solvent above the main phase transition temperature T , while agitation beyond two minutes did not appear to have an effect on the quality of the niosomes. One parameter which affected the quality of the niosomes was the loading of maltodextrin with surfactant. In the presence of excess surfactant, the maltodextrin surface becomes depleted and the dried film becomes unacceptably thick. Too little surfactant results in good quality niosomes, but the proportion of residual carrier would be higher. Good quality niosomes were easily obtained at 10-15 mM surfactant per gram of maltodextrin carrier, although higher loads (20-25 mM/g) could produce acceptable results. (8) At very high loading (>25 mM/g), adhesion of surfactant to carrier could not be maintained. Under these conditions, large surfactant fragments, which appeared to have been "cast" on spherical maltodextrin particles were observed. The resulting niosomes were harder to hydrate and were of very poor quality, similar to niosomes which were obtained from direct hydration of surfactant powder. Based on images of proniosomes with different surfactant loading (Figure 5), there appeared to be a correlation between thin, uniform coating and the quality of the niosomes. The advantage of using a carrier to increase the surface area is lost if the surfactant load results in a thick, irregular surface with a texture similar to that of surfactant powder.


m

Figure 5. Maltodextrin-based proniosomes. The surfactant loadings show here are 0.5 mmol/g (Ix), 2 mmol/g (4x), 4 mmol/g (8x), and 16 mmol/g (32x).

Proniosome and Hydrogel Powder Mixtures To extend the potential of this approach, a novel concept was tested in which proniosome powder was mixed with hydrogel powders such as agarose or gellan. The question in these experiments was whether the presence of the hydrogel polymer would compromise the ability of the surfactant to self-



46 assemble into niosomes. Similarly, it was possible that the presence of surfacetant would affect the gelling ability of the hydrogel polymer. To prepare the gels (Figure 6), proniosome powder and hydrogel powder were blended in a test tube by vortex mixing. Hot aqueous phase (buffer or water) was added while vortexing, and the tubes were maintained at high temperature in a heating block for several minutes. The temperature and the time of continued heating varied with the choice of hydrogel, and initial conditions were determined by manufacturer's recommendations. The tubes were removed frequently and vortexed to assure uniform hydration and swelling and to prevent settling.

Figure 6. Process for making niosome-containing hydrogels. The aqueo phase is added at elevated temperature (> T„), while the powder mixture being agitated. Continued heating of the mixture is required in order to ob gellation. In some cases, rapid cooling (i.e., on ice) is required in order t avoid settling. Niosomes formed in the hydrogel in all cases tested. Using light micro scopy, gel-entrapped proniosome-derived niosomes (GEProDeN) could be directly observed. Using a flourescent dye as a model "drug" allowed direct observation of the drug distribution of using fluorescence microscopy. For dyes with relatively good water solubility, fluorescence was observed in the niosomes and in the surrounding hydrogel. The relative distribution of dye appeared to be consistent with entrapment data determined for free niosomes. Poorly soluble fluorescent dye could be included in the formulation by adding the dye to the solution of surfactact in organic solvent. In this case, the dye was expected to be entrapped in the niosomes and not in the hydrogel. Fluorescence and brightfield microscopy showed that the presence of dye fluorescence correlated with the location of niosomes (Figure 7).



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Figure 7. Gel-entrapped-proniosome-derived-niosomes (GEProDeN) stain with Sudan 4 dye. This is an exposure with dual illumination, includi brightfield (green) illumination and epifluorescence (orange) illumination. method shows that the locations of the niosomes and the entrapped dye are correlated. (See page 1 of color insert.) Experiments were also performed with various types of ionic and zwitterionic lipids, including phospholipids. In all cases, proliposomes could be made from these lipids, and the proliposomes were suitable for forming multilamellar liposomes. In most cases, these proniosomes could be used to make gelentrapped-proliposome-derived-liposomes. Work is ongoing to evaluate appli cations for these formulations. Drug release from the hydrogel appears to occur by two mechanisms. Figure 8 shows that for soluble drug, the release is by both free diffusion of drug and by release of niosomes from the hydrogel matrix. For GEProDEN (Figure 8a), the release medium contains free drug in solution similar to that observed for drug-only hydrogels (Figure 8b) and a niosome suspension (as evidenced by light scattering) similar to that observed in control GEProDEN samples with no drug (Figure 8c).

Figure 8. Eosin Yellowish-release from GEProDEN (a) andfromhydrogel (b). A blank niosome-containing gel is shown in panel (c). The grid behind the samples is visible only in the sample which does not contain niosomes. Th images were obtained after 6 hours. (See page 1 of color insert.) These observations indicate that if the drug is soluble, direct diffusion of free drug from the hydrogel occurs with a timecourse similar to that observed with drug-only hydrogels. In addition, a later release of drug-containing nio somes occurs as the hydrogel begins to degrade. Some control over the release

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48 rates can be obtained by selection of the hydrogel material and concentration. The distribution of the drug between the niosomes and the hydrogel is passive, and is thus determined largely by the surfactant concentration in the formulation. For GEProDEN with poorly soluble drug, the only release mechanism is release of drug in niosomes as the hydrogel degrades. The concentation of free drug is negligible and the only significant drug is that associated with the hydro phobic domains of the niosomes. Figure 9 shows that after 6 hours, the drug is visible in the dissolution medium only when it is in the form of niosomes (Figure 9a). In control samples containing only free drug and hydrogel, there is no detectable absorbance characteristic of the drug in the dissolution medium.

Figure 9. GEProDEN containing Sudan 4, a poorly soluble drug. The drug is only released in niosomes (a), not asfreedrug (b). As seen in panels (a) and (c), light scattering due to release of niosomes obscures the grid behind th sample. (See page 1 ofcolor insert.)

Conclusions There are many possible drug delivery applications for niosomes or for GEProDEN. Advantages of proniosome-based or proliposome-based formu lations include the ability to solubilize poorly soluble actives, stability enhancement obtained through a dry formulation, which is hydrated only when needed, dose flexibility, and most of the advantages of conventional liposomal formulations. This proniosome formulation has also been tested using phosphorlipids, and multilamellar liposomes can be produced with this formulation. A large number of carrier powders have been tested, but the maltodextrin carrier is the best choice so far. Because this carrier is not soluble in chloroform, our organic solvent of choice, the process is very straightforward, and it is possible to scale up this method for bulk production. In its present stage of development, this approach appears to be best suited for the formulation of lipophilic drugs, especially in the case where these drugs are susceptible to hydrolysis. In vivo experiments are not yet underway, but to appreciate the full potential of the formulation, these experiments will be required in order to determine biodistribution and other properties. Similarly, further testing of the GEProDEN formulation will be required to evaluate its full potential.


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Acknowledgements


The authors wish to thank Adeniyi Fisayo, Mary Besl, Camille Persaud, Linh Pham, Phuong Tang, and Jose Cordero, who were all undergraduate research fellows with Dr. Rhodes and contributed to the niosome work. The original work of Chengjiu Hu is also gratefully acknowledged. Material contributions from Grain Products Corporation (Muscatine IA) were greatly appreciated.

References 1. Betageri, G.; Jenkins, S.; Parsons, D. Liposome Drug Delivery Systems Technomic Publishing Co. Inc., Lancaster, PA 1993. 2. Bangham, A.; Standish, M.; Watkins J. Mol. Biol. 1965, 13, 238-252. 3. Decher, G.; Ringsdorf, H.; Venzmer, J.; Bitter-Suermann, D.; Weisberger, C. Biochim. Biophys. Acta 1990, 1023, 357-364. 4. Herbette, L.; Rhodes, D.G.; Mason, R. Drug Design And Delivery 1991, 7, 75-118. 5. Yoshioka T.; Sternberg, B.; Florence, A. Int. J. Pharmaceutics 1994, 105, 1-6. 6. Hu,C.; Rhodes, D.G. Int. J. Pharmaceutics 1999, 185, 23 - 35. 7. Blazek-Welsh, A.I.; Rhodes, D. G. PharmSci 2001, 5, 1-8. 8. Blazek-Welsh, A.I.; Rhodes, D. G. Pharmaceutical Research 2001, 18, 656661.


Carrier-Based Drug Delivery - American Chemical Society

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