Formulation, development and assessment of efavirenz-loaded lipid nanocarriers

The feasibility of incorporating efavirenz (EFV) into innovative solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) using the hot high-pressure homogenization (HHPH) technique was investigated in an attempt to address the shortcomings in therapy associated with the use of conventional dosage forms. The shortcomings include the unpalatable taste of API in solution, instability in the presence of light when in solution and psychiatric side effects of the API. In particular, sustained release approaches may reduce or limit the incidence of adverse psychiatric effects of EFV and alleviate Acquired Immune Deficiency Syndrome (AIDS)-related complications such as AIDS Dementia Complex (ADC) in patients, ultimately improving their quality of life. Prior to initiating pre-formulation, formulation development and optimization studies of EFV-loaded SLN and/or NLC, Response Surface Methodology (RSM) in conjunction with central composite design (CCD), was used to develop and validate suitable methods for the quantitative determination of EFV in pharmaceutical formulations and for monitoring EFV release from SLN and/or NLC in vitro. Simple, accurate, precise, sensitive and stabilityindicating reversed phase-high performance liquid chromatography (RP-HPLC) methods with UV and electrochemical (EC) detection were developed, validated and optimized for in vitro analysis of EFV in formulations. On the basis of risk-to-benefit ratio the RP-HPLC method with UV detection was selected as the most suitable for the quantitative determination of EFV in pharmaceutical formulations, and was applied to in vitro release studies of EFV from SLN and/or NLC. Pre-formulation studies were undertaken to investigate the thermal stability of EFV so as to facilitate the selection of lipid excipients for the manufacture of nanocarriers, and to establish their compatibility with EFV. It was found that EFV was thermostable up to a temperature of approximately 200°C, indicating that HHPH could be used for the manufacture of EFV-loaded SLN and/or NLC. Lipid screening revealed that EFV is highly soluble in solid and liquid lipids, with glyceryl monostearate and Transcutol® HP showing the best solubilizing potential for EFV. Glyceryl monostearate exists in a stable β-modification prior to exposure to heat, but exists in the α-polymorphic modification following exposure to heat. It was established that the addition of Transcutol® HP to glyceryl monostearate revealed the co-existence of the α- and β’-polymorphic modifications, thereby revealing the existence of the modifications in NLC produced from the optimum lipid combination. Furthermore, an investigation of binary mixtures of EFV/glyceryl monostearate and glyceryl monostearate/Transcutol® HP, in addition to eutectic mixtures of EFV, glyceryl monostearate and Transcutol® HP, revealed no interaction between EFV and the lipids selected for the production of the nanocarriers. Due to the significantly higher solubility of EFV in Transcutol® HP than in to glyceryl monostearate, NLC are most likely to have a higher LC and EE than SLN. In addition, the existence of both the α- and β’-polymorphic modifications in the binary mixture of the lipid implies that EFV expulsion on prolonged storage is unlikely to occur from NLC when compared to SLN. Consequently formulation development and optimization studies of SLN and NLC were performed to investigate the potential to deliver EFV from a novel technology with an appropriate LC and EE for EFV. Tween®80 was selected for use in these formulations as the use of this surfactant facilitates the targeting of nanocarriers to the CNS. RSM in conjunction with a Box-Behnken Design (BBD) was used to establish the effects of process variables, such as number of homogenization cycles and pressure, in addition to formulation variables such as amount of EFV and Tween®80 on the particle size (PS), polydispersity index (PDI), zeta potential (ZP), visual assessment (VA) and release rate (RR) of EFV after 24 hours. In addition the LC and EE, degree of crystallinity and lipid modification, shape and surface morphology of the optimized batches were investigated to ensure that EFV-loaded SLN and NLC of desirable quality were produced. On the day of manufacture the mean PS and PDI of EFV-loaded SLN was 59.00 ± 23.16 nm and 0.382 ± 0.054 respectively. The mean PS and PDI of EFV-loaded NLC was 34.73 ± 0.7709 nm and 0.394 ± 0.027 respectively. The formulations were in the nanometer range and exhibited a narrow particle size distribution, as indicated by the PDI values. The ZP values for optimized SLN and NLC generated on the day of manufacture using HPLC grade water as the dispersion medium were -32.5 ± 4.99 mV and -22.4 ± 3.72 mV respectively. In addition the optimized batches of SLN and NLC revealed a decrease in crystallinity in comparison to bulk lipid material. DSC, WAXS and FT-IR revealed that EFV was molecularly dispersed in the nanocarriers. In addition EFV-loaded SLN existed in a single α-polymorphic form, whereas EFV-loaded NLC exhibited the co-existence of α- and β’-polymorphic forms. Generally SLN and NLC were spherically shaped when viewed under transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On the day of manufacture the EE and LC of EFVloaded SLN was found to be 96.77 ± 0.453 % and 9.68 ± 1.772 % respectively. The EE and LC of EFV-loaded NLC was 99.93 ± 0.413 and 9.995 ± 0.672 respectively. The release profiles for the optimized formulations of SLN and NLC exhibited an initial burst release over the first 0-3 hours of testing, after which the release was sustained for up to 24 hours. The cumulative % EFV released over 24 hours was higher from SLN (91.5±3.423 %) than that observed for NLC (73.6±4.34 %). Stability studies performed for 8 weeks on the optimized batches of the SLN and the NLC were also conducted so as to ensure product quality. The formulations were assessed in terms of parameters considered benchmarks of stability, and included ZP, PS, PDI, LC and EE. Generally these parameters remained unchanged following storage for 8 weeks at 25°C/60% RH but showed considerable changes following storage for 8 weeks at 40°C/75% RH. These studies reveal that SLN and NLC when stored at 25°C/60% RH have the potential to be used as colloidal delivery systems for EFV that have the potential to protect EFV from photodegradation and sustain release into brain tissue. The latter will ultimately reduce or limit the incidence of adverse psychiatric effects and potentially alleviate AIDS-related complications such as ADC in patients with HIV/AIDS, ultimately improving their quality of life.Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 201

Lipid nanocarriers : a novel approach to delivering ophthalmic clarithromycin

The feasibility of incorporating clarithromycin (CLA) into innovative solid lipid nanoparticles (SLN) and nanostructured lipi d carriers (NLC) using hot emulsification ultrasonication (HEUS) was investigated. This approach was investigated in an attempt to address the shortcomings associated with the use of lyophilized parenteral formulations administered via the ocular route suc h as toxic reactions, intolerance and patient discomfort due to frequent insti llation of topical solutions of CLA. In particular, sustained release approaches to delivery may enhance precorneal retention, increase ocular availability and permit dose reduction or use of a longer dosing frequency when treating ocular non - tuberculous m ycobacterial (NTM) keratitis infections. This approach may potentially improve the delivery of CLA to the eye, thereby addressing some or all of the unmet clinical needs described vide infra . Prior to initiating pre - formulation, formulation development a nd optimization studies of CLA - loaded SLN and/or NLC, Design of Experiments (DoE), specifically a Central Composite Design (CCD) was used in conjunction with Response Surface Methodology (RSM) to develop and optimize a suitable method for the quantitative determination of CLA in pharmaceutical formulations and for monitoring CLA release from SLN and/or NLC in vitro . A simple, accurate, precise, sensitive and stability - indicating reversed phase - high performance liquid chromatography (RP - HPLC) method with ele ctrochemical (EC) detection was developed, validated and optimized for the in vitro analysis of CLA loaded SLN and/or NLC formulations. Pre - formulation studies were undertaken to investigate the thermal stability of CLA and bulk lipids to facilitate the s election of lipid excipients for the manufacture of nanocarriers in addition to establishing compatibility of CLA with the excipients. It was established that CLA was thermostable up to a temperature of approximately 300 °C thereby indicating that HEUS cou ld be used for the manufacture of CLA - loaded SLN and/or NLC. Lipid screening revealed that CLA i s, in general, poorly soluble in solid and liquid lipids however a combination of stearic acid (SA) and Transcutol ® HP (THP) exhibited the best dissolution pote ntial for CLA of all lipids tested . Stearic acid appears to exist as polymorphic form B prior to exposure to heat however occurs as the form C polymorph following heating at 85 °C for one hour. The best ratio for the mixture of SA and THP for the manufactu re of CLA - NLC ii was an 80:20 ( w/w ) ratio of SA: THP as the two lipids are miscible in this ratio and exhibited the greatest dissolution potential for CLA. Furthermore, an investigation of binary mixtures of CLA/SA and SA/Transcutol ® HP, in addition to eutect ic mixtures of CLA, SA and Transcutol ® HP, revealed no obvious interaction between CLA and the lipids selected for the production of the nanocarriers. Due to the relatively high solubility of CLA in THP in comparison to SA, NLC are likely to exhibit a hig her loading capacity (LC) and encapsulation efficiency (EE) for CLA than SLN. Consequently the feasibility of incorporating CLA (10% w/w ) into NLC was investigated and evaluation of the production of SLN was not undertaken as the production of these might not result in the manufacture of a delivery technology with a high EE and LC for CLA. Tween ® 20 was used as the surfactant as it is readily available, exhibits little or no cytotoxicity and is relatively cheap. Polyethylene glycol (PEG) was used as a coati ng polymer to impart muco - adhesive properties the formulated CLA - NLC. Response surface methodology (RSM) in conjunction with DoE, specifically a Box - Behnken Design (BBD) used as a screening design was used to identify a formulation composition which would produce a product that would meet the pre - defined target critical quality attributes (CQA) for the nanoparticles viz. particle size (PS) in the nano - range, polydispersity index (PDI) 80%. The formulation composition identified was subsequently used for the optimization of the manufacturing parameters viz. sonication time and amplitude, using a Central Composite Design (CCD) . The LC and EE, in vitro CLA release, cytotoxicity, osmolarity, pH, degree of crystallinity and lipid modification, elemental analysis and surface morphology of the optimized batch was investigated and mon itored to ensure that CLA - loaded NLC, of the desirable quality, had been produced. On the day of manufacture the mean PS and PDI of the optimized CLA - loaded NLC formulation adjusted to physiological osmolarity (250 – 450 mOsm/kg) was 461.9 ± 40.16 nm and 0. 523 ± 0.104, respectively. The ZP for the optimized NLC generated on the day of manufacture using HPLC grade water as the dispersion medium was - 20.5 ± 4.82 mV. The pH and osmolarity of the optimized CLA - loaded NLC formulation was 7.76 ± 0.01 and 316 ± iii 2 m Osm/Kg, respectively and the EE was 88.62 ± 0.23 %. The optimized NLC exhibited a decreased crystallinity in comparison to the bulk lipid materials. DSC, WAXS and FT - IR revealed that CLA was molecularly dispersed in the nanocarriers. The optimized CLA - load ed NLC exhibited muco - adhesive properties, when tested under stationary conditions using laser doppler anemometry (LDA). The optimized formulation also exhibited sustained release of CLA over 24 hours during in vitro release testing and CLA release was bes t described using the Baker - Lonsdale model . The cumulative % CLA released over 24 hours was 56.13 ± 0.23% and mass balance analysis revealed 41.38 ± 0.02% CLA had been retained in the NLC. In vitro cytotoxicity testing revealed that the optimized CLA - NLC w ere less cytotoxic to HeLa cells when compared to CLA alone and further confirmed that the lipids and excipients used in these studies were of GRAS status . Stability studies revealed that the EE reduced over 28 days by 14.42% and 5.14% when stored at 4 °C and 22 °C , respectively. In addition, the particle size increased from the nm to μm range for samples stored at 22 °C. The findings are a good starting point but require further optimization to ensure prolongation of stability. In addition , the technology requires additional developmental studies and a powder for reconstitution for use as a single - dose considered as single dose packaging may be a solution to the compromised formulation stability observed in these studies. The CLA - NLC produced in these stu dies exhibit sound product attributes which serve as a useful foundation for the novel delivery of antibiotics to the eye. The results suggest that the optimized NLC have the potential to enhance precorneal retention and increase ocular availability of CLA , which in turn may be useful to reduce the required dose and dosing frequency when administering CLA as a reconstituted solution to treat susceptible organisms that infect ocular tissues

Enhancement of Biological and Pharmacological Properties of an Encapsulated Polyphenol: Curcumin.

There is a dearth of natural remedies available for the treatment of an increasing number of diseases facing mankind. Natural products may provide an opportunity to produce formulations and therapeutic solutions to address this shortage. Curcumin (CUR), diferuloylmethane; I,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is the major pigment in turmeric powder which has been reported to exhibit a number of health benefits including, antibacterial, antiviral, anti-cancer, anti-inflammatory and anti-oxidant properties. In this review, the authors attempt to highlight the biological and pharmacological properties of CUR in addition to emphasizing aspects relating to the biosynthesis, encapsulation and therapeutic effects of the compound. The information contained in this review was generated by considering published information in which evidence of enhanced biological and pharmacological properties of nano-encapsulated CUR was reported. CUR has contributed to a significant improvement in melanoma, breast, lung, gastro-intestinal, and genito-urinary cancer therapy. We highlight the impact of nano-encapsulated CUR for efficient inhibition of cell proliferation, even at low concentrations compared to the free CUR when considering anti-proliferation. Furthermore nano-encapsulated CUR exhibited bioactive properties, exerted cytotoxic and anti-oxidant effects by acting on endogenous and cholinergic anti-oxidant systems. CUR was reported to block Hepatitis C virus (HCV) entry into hepatic cells, inhibit MRSA proliferation, enhance wound healing and reduce bacterial load. Nano-encapsulated CUR has also shown bioactive properties when acting on antioxidant systems (endogenous and cholinergic). Future research is necessary and must focus on investigation of encapsulated CUR nano-particles in different models of human pathology

Formulation, development and assessment of efavirenz-loaded lipid nanocarriers

The feasibility of incorporating efavirenz (EFV) into innovative solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) using the hot high-pressure homogenization (HHPH) technique was investigated in an attempt to address the shortcomings in therapy associated with the use of conventional dosage forms. The shortcomings include the unpalatable taste of API in solution, instability in the presence of light when in solution and psychiatric side effects of the API. In particular, sustained release approaches may reduce or limit the incidence of adverse psychiatric effects of EFV and alleviate Acquired Immune Deficiency Syndrome (AIDS)-related complications such as AIDS Dementia Complex (ADC) in patients, ultimately improving their quality of life. Prior to initiating pre-formulation, formulation development and optimization studies of EFV-loaded SLN and/or NLC, Response Surface Methodology (RSM) in conjunction with central composite design (CCD), was used to develop and validate suitable methods for the quantitative determination of EFV in pharmaceutical formulations and for monitoring EFV release from SLN and/or NLC in vitro. Simple, accurate, precise, sensitive and stabilityindicating reversed phase-high performance liquid chromatography (RP-HPLC) methods with UV and electrochemical (EC) detection were developed, validated and optimized for in vitro analysis of EFV in formulations. On the basis of risk-to-benefit ratio the RP-HPLC method with UV detection was selected as the most suitable for the quantitative determination of EFV in pharmaceutical formulations, and was applied to in vitro release studies of EFV from SLN and/or NLC. Pre-formulation studies were undertaken to investigate the thermal stability of EFV so as to facilitate the selection of lipid excipients for the manufacture of nanocarriers, and to establish their compatibility with EFV. It was found that EFV was thermostable up to a temperature of approximately 200°C, indicating that HHPH could be used for the manufacture of EFV-loaded SLN and/or NLC. Lipid screening revealed that EFV is highly soluble in solid and liquid lipids, with glyceryl monostearate and Transcutol® HP showing the best solubilizing potential for EFV. Glyceryl monostearate exists in a stable β-modification prior to exposure to heat, but exists in the α-polymorphic modification following exposure to heat. It was established that the addition of Transcutol® HP to glyceryl monostearate revealed the co-existence of the α- and β’-polymorphic modifications, thereby revealing the existence of the modifications in NLC produced from the optimum lipid combination. Furthermore, an investigation of binary mixtures of EFV/glyceryl monostearate and glyceryl monostearate/Transcutol® HP, in addition to eutectic mixtures of EFV, glyceryl monostearate and Transcutol® HP, revealed no interaction between EFV and the lipids selected for the production of the nanocarriers. Due to the significantly higher solubility of EFV in Transcutol® HP than in to glyceryl monostearate, NLC are most likely to have a higher LC and EE than SLN. In addition, the existence of both the α- and β’-polymorphic modifications in the binary mixture of the lipid implies that EFV expulsion on prolonged storage is unlikely to occur from NLC when compared to SLN. Consequently formulation development and optimization studies of SLN and NLC were performed to investigate the potential to deliver EFV from a novel technology with an appropriate LC and EE for EFV. Tween®80 was selected for use in these formulations as the use of this surfactant facilitates the targeting of nanocarriers to the CNS. RSM in conjunction with a Box-Behnken Design (BBD) was used to establish the effects of process variables, such as number of homogenization cycles and pressure, in addition to formulation variables such as amount of EFV and Tween®80 on the particle size (PS), polydispersity index (PDI), zeta potential (ZP), visual assessment (VA) and release rate (RR) of EFV after 24 hours. In addition the LC and EE, degree of crystallinity and lipid modification, shape and surface morphology of the optimized batches were investigated to ensure that EFV-loaded SLN and NLC of desirable quality were produced. On the day of manufacture the mean PS and PDI of EFV-loaded SLN was 59.00 ± 23.16 nm and 0.382 ± 0.054 respectively. The mean PS and PDI of EFV-loaded NLC was 34.73 ± 0.7709 nm and 0.394 ± 0.027 respectively. The formulations were in the nanometer range and exhibited a narrow particle size distribution, as indicated by the PDI values. The ZP values for optimized SLN and NLC generated on the day of manufacture using HPLC grade water as the dispersion medium were -32.5 ± 4.99 mV and -22.4 ± 3.72 mV respectively. In addition the optimized batches of SLN and NLC revealed a decrease in crystallinity in comparison to bulk lipid material. DSC, WAXS and FT-IR revealed that EFV was molecularly dispersed in the nanocarriers. In addition EFV-loaded SLN existed in a single α-polymorphic form, whereas EFV-loaded NLC exhibited the co-existence of α- and β’-polymorphic forms. Generally SLN and NLC were spherically shaped when viewed under transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On the day of manufacture the EE and LC of EFVloaded SLN was found to be 96.77 ± 0.453 % and 9.68 ± 1.772 % respectively. The EE and LC of EFV-loaded NLC was 99.93 ± 0.413 and 9.995 ± 0.672 respectively. The release profiles for the optimized formulations of SLN and NLC exhibited an initial burst release over the first 0-3 hours of testing, after which the release was sustained for up to 24 hours. The cumulative % EFV released over 24 hours was higher from SLN (91.5±3.423 %) than that observed for NLC (73.6±4.34 %). Stability studies performed for 8 weeks on the optimized batches of the SLN and the NLC were also conducted so as to ensure product quality. The formulations were assessed in terms of parameters considered benchmarks of stability, and included ZP, PS, PDI, LC and EE. Generally these parameters remained unchanged following storage for 8 weeks at 25°C/60% RH but showed considerable changes following storage for 8 weeks at 40°C/75% RH. These studies reveal that SLN and NLC when stored at 25°C/60% RH have the potential to be used as colloidal delivery systems for EFV that have the potential to protect EFV from photodegradation and sustain release into brain tissue. The latter will ultimately reduce or limit the incidence of adverse psychiatric effects and potentially alleviate AIDS-related complications such as ADC in patients with HIV/AIDS, ultimately improving their quality of life.Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 201

Applications of Electrospun Drug-Eluting Nanofibers in Wound Healing: Current and Future Perspectives

Wounds are a consequence of disruption in the structure, integrity, or function of the skin or tissue. Once a wound is formed following mechanical or chemical damage, the process of wound healing is initiated, which involves a series of chemical signaling and cellular mechanisms that lead to regeneration and/or repair. Disruption in the healing process may result in complications; therefore, interventions to accelerate wound healing are essential. In addition to mechanical support provided by sutures and traditional wound dressings, therapeutic agents play a major role in accelerating wound healing. The medicines known to improve the rate and extent of wound healing include antibacterial, anti-inflammatory, and proliferation enhancing agents. Nonetheless, the development of these agents into eluting nanofibers presents the possibility of fabricating wound dressings and sutures that provide mechanical support with the added advantage of local delivery of therapeutic agents to the site of injury. Herein, the process of wound healing, complications of wound healing, and current practices in wound healing acceleration are highlighted. Furthermore, the potential role of drug-eluting nanofibers in wound management is discussed, and lastly, the economic implications of wounds as well as future perspectives in applying fiber electrospinning in the design of wound dressings and sutures are considered and reported

Lipid-Based Nanocarriers for Neurological Disorders: A Review of the State-of-the-Art and Therapeutic Success to Date.

Neurodegenerative disorders including Alzheimer's, Parkinson's, and dementia are chronic and advanced diseases that are associated with loss of neurons and other related pathologies. Furthermore, these disorders involve structural and functional defections of the blood-brain barrier (BBB). Consequently, advances in medicines and therapeutics have led to a better appreciation of various pathways associated with the development of neurodegenerative disorders, thus focusing on drug discovery and research for targeted drug therapy to the central nervous system (CNS). Although the BBB functions as a shield to prevent toxins in the blood from reaching the brain, drug delivery to the CNS is hindered by its presence. Owing to this, various formulation approaches, including the use of lipid-based nanocarriers, have been proposed to address shortcomings related to BBB permeation in CNS-targeted therapy, thus showing the potential of these carriers for translation into clinical use. Nevertheless, to date, none of these nanocarriers has been granted market authorization following the successful completion of all stages of clinical trials. While the aforementioned benefits of using lipid-based carriers underscores the need to fast-track their translational development into clinical practice, technological advances need to be initiated to achieve appropriate capacity for scale-up and the production of affordable dosage forms