SOLID LIPID NANOPARTICLES

Solid Lipid Nanoparticles for the Delivery of Pharmaceutical Actives By: Andrew Loxley, PhD

INTRODUCTION An increasing number of active pharmaceutical ingredients (APIs) under development are poorly water soluble and therefore have poor bioavailability. These are designated Biopharmaceutical Classification System (BCS) class II and class IV APIs.1-3 Creative formulation efforts are required to produce a finished drug product from these APIs that has acceptable pharmacokinetics. A common formulation approach with such compounds is to focus on creating and stabilizing very small particles of the API in an attempt to increase the surface area available for dissolution in vivo, and hence the rate of dissolution, and consequently plasma or tissue levels of API. Another approach is to create so-called solid solutions of the API.4 Biologics (proteins, peptides, oligonucleotides, and SiRNAs) are water soluble but bring their own formulation and delivery challenges. Shelf-life stability and enzymatic degradation are two main areas of concern, and formulation design focuses on stabilizing the API in storage and protecting it from endogenous enzymes until it reaches its therapeutic target. In more advanced formulations, the API is formulated into a delivery vehicle that specifically targets tissue or cells to maximize the therapeutic index.

NANOPARTICLE core of the vesicles, or as molecularly Issues with shelf-life stability of the FORMULATIONS dissolved material in the lipid bilayer.6 finished product or the need for organic Biodegradable polymers have also been solvents in processing for many of these Many of the aforementioned used to form API-loaded nanoparticles or approaches render them less than ideal. formulation approaches utilize block copolymer micelles or polymersomes, nanotechnology, that is, the preparation of usually by emulsification/solvent-

7,8

Vol 9 No 8 sub-micron structures containing the API. evaporation techniques. Biocompatible For BCS class II and IV APIs, the simplest and biodegradable inorganic nanoparticles F I G U R E 1 nanoparticle is made of pure API, formed can be loaded with API via a by top-down processes starting with bulk microemulsion technique.9 Biologics and API, such as milling, grinding, other water-soluble drugs have been September 2009 homogenization, ultrasonication, and are incorporated into the aqueous core of stabilized in dispersion by the presence of a liposomes, into the aqueous domains of surfactant.5 Alternatively, bottom-up “self- biodegradable polymer nanoparticles assembly” processes can be used, such as prepared by water-in-oil-in-water anti-solvent precipitation and micellar emulsion/solvent evaporation, and charge- incorporation by dilution. For example, neutralization nano-complexes made by

Drug Delivery Technology insoluble APIs may be incorporated into interaction with oppositely charged xx nano-sized vesicles or liposomes, in the polyelectrolyes, or by attachment to gold form of particles dispersed in the aqueous nanoparticles.10-13 SOLID LIPID NANOPARTICLES

SOLIDLIPIDNANOPARTICLES– F I G U R E 2 MATERIALS & SYNTHESIS

Many biocompatible/biodegradable lipids are solid at room temperature, can be obtained in high purity, are generally recognized as safe (GRAS), and are inexpensive. Some common solid lipids used to make solid lipid nanoparticles (SLNs) include triglycerides (eg, Compritol 888 ATO and Dynasan 112), carnauba wax, beeswax, cetyl alcohol, emulsifying wax, cholesterol, and cholesterol butyrate. Nano- and microparticles made of these lipids and suspended in water offer an option for formulating both BCS class II and IV APIs as well as biologics that may overcome the issues of shelf-life stability and the cost and toxicity associated with the use of organic solvents. In effect, the concepts of nanoparticles and solid solutions are being combined. Nanoparticles of these lipids may be made F I G U R E 3 using a templated synthesis from a microemulsion of the molten lipid in aqueous surfactants, by precipitation of the wax from a solution in a non-ionic surfactant on addition of water, or by emulsifying the molten lipid into a hot aqueous surfactant solution with high-shear mixing to obtain the desired submicron particle size.14-16

API ENCAPSULATION IN SLNs Vol 9 No 8 Small molecules can be entrapped within the lipid matrix of the nanoparticles by dissolving or dispersing the material in the molten lipid prior to particle formation. Souto’s

PhD thesis on the delivery of APIs using SLNs September 2009 lists more than 100 APIs that have been encapsulated in SLNs.17 An SEM of the particles of a typical SLN dispersion (in this case of particles containing the sunscreen octylmethoxycinnamate) is

shown in Figure 1. Particles of this type are Drug Delivery Technology made at commercial scale for formulation into XX SOLID LIPID NANOPARTICLES

F I G U R E 4 aerosol showed that the particle size distribution of the SLNs in the original dispersion was maintained in the droplets. The droplet size of the aerosol was also found to be ideal for delivery to the deep lung (around 5 microns).This work could lead to improved pulmonary delivery of water-insoluble APIs for acute treatment in hospitals where doses may need to be high.

SURFACE ENTRAPMENT OF APIs WITH SLNs

Instead of incorporating the drug into the particle, an additional way to exploit SLNs is to attach the API to the surface of the particle. The surface properties of SLNs can be varied widely and tailored to the final application. For example, the choice of emulsifier (cationic, non-ionic, anionic, and polymeric) has a strong influence on the surface electrical charge on the nanoparticles, measured by the zeta potential of the particles, as shown in Figure 3. topical products to provide UV-protection. tetramethylindocarbocyanine perchlorate (DiI) For SLNs that contain long-chain fatty In some cases, the API is not compatible by a modified preparation technique to acids or use them as the emulsifier, the with the lipid and is expelled from the accommodate the low solubility of DiI in the carboxyl groups present at the particle surface nanoparticle, usually during cooling and molten lipid. Fluorescent SLNs are useful to can be used to covalently attach proteins and solidification. This can lead to undesirable follow the fate of particles applied mucosally amine-terminated peptides using standard macroscopic crystals of API in the final in vivo and determining efficiency of uptake coupling chemistries (such as carbodiimide formulation or phase separation of the by antigen presenting cells in vitro in the coupling). particles to structures as complex as “nano- development of a novel HIV vaccine.20 Tissue Biologics are generally charged in spoons.”18 By mixing liquid lipids with the samples taken from penile epithelial explants aqueous solution, and as such are attracted solid lipid prior to particle formulation, lipid after application of fluorescent SLNs show electrostatically to surfaces of opposite charge, crystallization is hindered or prevented, and a particles penetrated well into the tissue and may become strongly attached there as a Vol 9 No 8 more amorphous nanoparticle internal (Figure 2A), and dendritic cells are shown to result. We have found that electrically charged structure is achieved. In this way, APIs are less internalize green fluorescent SLNs following SLNs (cationic or anionic) strongly and likely to be expelled from SLNs during the incubation in vitro (Figure 2B). irreversibly bind proteins with attachment cooling step of their preparation, and stable The green fluorescent particles were also efficiencies of around 90% (around 650

September 2009 SLNs may be formulated with a wider range used in a proof-of-concept study for the micrograms protein per mg of SLN solids). of APIs.19 development of an inhalable API-loaded SLN Evidence that the protein is attached at the As one example created at Particle dispersion. A fluorescent dye-loaded SLN particle surface is provided by the observation Sciences, fluorescent SLNs have been dispersion was aerosolized using an OTC that after mixing the SLN and protein and prepared by adding a fluorescent dye to the nebulizer, and the aerosol plume from the allowing enough time for the protein to adsorb molten lipid prior to particle preparation. mouthpiece was illuminated by UV light. The at the particle surface, the pH-dependence of

Drug Delivery Technology Green fluorescent SLNs were prepared with green fluorescent glow of the plume showed the SLN’s zeta-potential goes from that of the pyrromethene 567A dye, and red fluorescent that the SLNs were indeed in the aerosol naked SLN to that of the pure protein. XX SLNs with 1,1’-dioctadecyl-3,3,3’,3’- droplets, and analysis of the condensed SOLID LIPID NANOPARTICLES

Essentially the SLNs surface properties F I G U R E 5 become dominated by the protein attached there (Figure 4). Based on the encouraging tissue and cellular uptake results and the ability to efficiently and simply attach proteins to the SLN surface, nanoparticles made of carnauba wax and formulated to carry gp140 (a model HIV antigen) were applied to the vaginal mucosa of mice to evaluate this route of administration as a novel approach to vaccination against HIV. As controls in this experiment, mice were also vaccinated by subcutaneous injection of the SLN-gp140 formulation as well as a formulation using alum, the only particles used in generally approved particle-containing vaccines in the US. The systemic challenge results with the SLNs were equivalent to the alum control (data not shown), indicating that these particular SLNs are potentially promising adjuvants for systemic vaccination.

STERILIZATION

For parenteral administration, SLN dispersions must be sterile. The mean particle diameter of SLNs is often more than 200 nm, so sterile filtration is not possible in these cases. Autoclaving the finished dispersion is not practical as the lipids melt at temperatures used to terminally heat-sterilize pharmaceutical products, and the molten lipid droplets coalesce as there is no applied shear to prevent this. Options are therefore limited Vol 9 No 8 to aseptic manufacturing processes following sterilization of the starting materials (gamma or e-beam irradiation of the final dispersion) or exposure to ethylene oxide gas (EO).

Bacterial endotoxins in raw materials need to September 2009 be monitored, especially when raw materials are of natural origin. It may be possible to lyophilize the SLN dispersion, and this lyophile can be irradiated or exposed to EO. We have demonstrated that lyophilized SLNs

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D g ru D s hy p io B elivery. d . g g ru d scatterin t h lig laser elea R le g l ltian . tro u 3 n 7 m o 8 C J lved e-reso atite. ap tim y rox d 2 hy . d 8 cta. 4 A an -1 2 s 4 A hy ):1 p G L (2 io 8 P B 2 ;1 g 8 0 in im 0 2 ch tain io n B co d o ects. eth asp m tical aceu arm h p io b a: m am g REFERENCES i / 3179695. 5 9 6 9 7 1 I3 A s/A n tio pt pdf. d .p 0 5 0 2 0 8 t0 /p 0 5 0 2 0 8 otc do lht l. tm il.h ox /d tech io ctonoft op200oral o 0 0 2 p to e th f o n icatio oahe oi enai al n atio tern in to es ach ro p p corf umordietd irected d r o m tu r fo r ecto ctoni ugdelivery. d g ru d in n icatio clJ 80( : - 4 6 ):8 (2 0 ;8 1 0 0 2 J. ical 96. 6 -9 5 8 hAnnua ei & g eetin M al u n n A th 0 3 s tntNo. o N t aten P S U es. pe 3179695. 5 9 6 9 7 1 I3 A A er ap sf nvir ma ng g in ag im itro v in r fo es a n.MolPham. arm h P l o M . an Jap d ncr Jnuay1, 1 ary u (Jan cer. an poons sonsa d an s n isio v s. n o o sp o n ae e nd d an ce, valen ce nduc tongi in g n stro ce u d in icles e or tonwih ith w n atio rm fo lex p . 2 1 el.Na ett. L o an N cells. ie : sited tce.USPae . o N t aten P S U rticles. ni efr - n terfero in an m u h r ul 23, 3 -2 9 1 ly Ju . 1 8 1 ndprvii al n isio rov p d an s g ss e ree F esis, h T D h P . tntNo. o N t aten P S U 21( : 422. 2 2 -4 7 8 ):3 (5 1 ;2 1530: 145. 5 4 -1 4 3 :1 0 3 5 ;1 1 0 0 se. We son n lsio u em /W lvr he e th : elivery d g ee e at ted resen p Bristol. of University the from microencapsulation on in focusing PhD Chemistry Physical his and Sussex from of Chemistry Univeristy in the BSc his earned He Ltd. Synthomer at adhesives latex-based and Corp., EINK at Inc, displays Systems electrophoretic A123 at batteries ion lithium next-generation in efforts development the led he Sciences, Particles joining to Prior delivery. 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