METHOD FOR ENCAPSULATING PHARMACEUTICAL ACTIVES
This invention relates to the encapsulation of pharmaceutical actives. In
particular, the invention relates to a method for encapsulating a pharmaceutical active.
The method is capable of producing pharmaceutical active-loaded particles (i.e. drugloaded
particles) suitable for the production of nano and/or micro pharmaceutical
formulations.
Field of the Invention
This present invention thus lies in the field of drug delivery/pharmaceutical
formulations and particularly relates to a distinctive encapsulation method or technique
that can be described as an oieo-po!ymeric method for encapsulating pharmaceutical
actives or drugs with either hydrophilic or hydrophobic properties. The oleo-polymeric
method of the invention advantageously can produce nano/micro pharmaceutical
formulations that can be administered via the oral or transmucosal routes.
Background to the Invention
Encapsulation is widely applied in pharmaceutical formulation production
for entrapping essential or active ingredients into an external carrier system in order to
impart protection against oxidation, degradation and isomerization as well as enhance
stability, improve functionality and extend product shelf life. Furthermore, encapsulation
of a bioactive can be utilized for controlling the release of bioactives under both in vitro
and in vivo conditions. Nano/micro encapsulation or fabrication refers to the tools and
methods that can be used for modifying the structures and properties of a
substance/material at the nano scale (< 1000 nm) or micro scale (< 100 m).
In this specification, the term "pharmaceutical active" is intended to refer to
an active pharmaceutical ingredient, also known as an API.
Advancements in the production of nanomedicines possess great potential
for reforming pharmacotherapeutical processes. Nano/micro-range drug delivery
systems constitute an important part of nanomedicine. The development of useful
pharmaceuticals based on the principles of nanomedicine can be described as the
science and technology of composite systems within the nanometer scale { -1000 nm)
consisting of two or more constituents which are the drug moiecule(s) and excipient(s).
A nano-formulation consisting of only the pulverized pharmaceutically active molecule is
also possible. Defining nanotechnology in drug delivery by placing limits on size is not
always helpful because the usefulness of such systems is typically not based on a size
limit. In a nutshell, useful drug delivery systems span over accurately nano-sized (<
000 nm) to micro-sized systems ( 100 ) and have been useful for developing
clinically effective formulations. From a real-world perspective, based on these
reasons, nanotechnology includes microtechnology and nanofabrication or
nanomanufacturing and its micro counterparts.
Research has proven that nano/micro encapsulation is an idea! way of
preparing controlled, rate-modulated pharmaceutical formulations that are beneficial to
patients. Nano/micro formulations have numerous unique advantages which include
but are not limited to the following: (i) delivery of drug molecules with poor solubility and
permeability, (ii) attainment of constant plasma drug concentration and increased
relative bioavailability, (iii) site-specific targeted drug delivery, (iii) immediate release
properties for rapid onset of pharmacological action, (iv) formulation taste improvement,
(v) sustained release leading to reduction of dosage frequency as well as both local and
systemic side effects, (vi) enhanced mucoadhesion and transmucosal permeation (vii)
good stability suitable for extended storage duration with little or no loss of drug activity,
(viii) enables administration of formulations (e.g. effervescent dosage forms) to patients
who are unable to chew or swallow, (ix) increased formulation morphological flexibility
and surface area; x) less dose of active drug is required, (xi) decreased fasted or fed
erraticism and (xii) reduced patient-to-patient variability.
Several methods have been developed for the preparation of a variety of
polymeric nano/micro-products. This can be divided into two main groups namely
dispersion of preformed polymers and polymerization of monomeric units. The
dispersion of preformed polymers encompasses processes such as evaporation or
extraction of solvent, nanoprecipitation, emulsification/solvent diffusion, salting out,
dialysis, supercritical fluid technology and hot melt encapsulation. Preparation methods
which involve monomeric units include techniques such as emulsion, mini/micro
emulsion, interracial, surfactant-free emulsion, controlled/living radical polymerizations
and interfacial polycondensation.
Currently, the emulsion technique, either using monomers or preformed
polymers dispersed in a mixed aqueous and/or non-aqueous continuous phase, is one
of the quickest, easily scalable, most commonly employed methods of preparing
nano/micro formulations. It presents several advantages such as batch-to-batch
reproducibility, simplicity and narrow size distribution as well as relatively high
encapsulation efficiency. Its major disadvantages include the use of toxic organic
solvents, harsh/varying temperature, pressure and pH conditions as weil as leakage of
encapsulated drug molecules, especially hydrophilic-natured ones, into the aqueousorganic
external phase thereby reducing encapsulation efficiency/drug loading capacity.
In other words, this method is more suitable for hydrophobic drugs indicating its nonversatility.
The vast advantages of nano/micro formulations and the demerits of the
choice of emulsion technique make developing a unique technology which can produce
pharmaceutically useful nano/micro formulations for commercialization processes and
which overcomes the drawbacks of the prior art desirable.
According to the invention, there is provided a method for encapsulating a
pharmaceutical active, the method including
dispersing a matrix former into at least one of a water phase and an oil phase,
with the pharmaceutical active being present in at least one of the oil phase and the
water phase and with the oil phase comprising a vegetable-based edible oil;
forming an emulsion from the oil phase and the water phase; and
lyophilizing the emulsion to form a solid lyophilisate.
Preferably, the emulsion is a finely dispersed emulsion. More preferably,
the emulsion is so finely dispersed that it appears mono-phased and homogenous, so
that it can be referred to as a nano/micro-emulsion.
The method typically includes comminuting the solid lyophilisate to provide
particles or a powder of the encapsulated pharmaceutical active. Preferably, the
powder is suitable for the production of nano and/or micro pharmaceutical formulations.
The invention thus also extends to a method of producing nano and/or micro
pharmaceutical formulations.
Comminuting the solid lyophilisate may include dry milling. Preferably, a
free flowing powder is provided by comminution. Preferably, particles of the powder are
spherical. The powder is typically a powder of a semi-crystalline solid material.
The matrix former may be a polymeric matrix former. Instead, the matrix
former may be a non-polymeric matrix former.
The matrix former may be hydrophilic. Instead, the matrix former may be
hydrophobic.
In one embodiment of the invention, a hydrophilic matrix former s
dispersed into the water phase and a hydrophobic matrix former is dispersed into the oil
phase.
The hydrophilic matrix former may be a polymeric matrix former.
The hydrophilic polymeric matrix former may be selected from the group
consisting of a polyethylene glycol, polyacrylic acid, polyether, polyacrylamide, polyvinyl
alcohol, polyethylene oxide, polyvinylpyrrolidone, polyoxazo!ine, po!ymethacrylate,
polyethylenimine, polyphosphate based polymer and mixtures of two or more thereof.
The hydrophobic matrix former may be selected from the group consisting
of a cellulose ether, a poly (lacttde-co-glycolide) based polymer, and mixtures of two or
more thereof.
In an exemplified embodiment of the invention, the hydrophilic matrix
former is polyethylene glycol and the hydrophobic matrix former is ethyl cellulose.
The process may include employing a water soluble polymer as a
surfactant or emulsifying agent for the forming of an emulsion, preferably finely
dispersed emulsion, from the oil phase and the water phase. In one embodiment of the
invention, the water soluble polymer is added to the water phase.
The water soluble polymer may be selected from the group consisting of
polyvinyl alcohols, methylcellulose derivatives, cross linked copolymers of acrylic acid,
and mixtures of two or more thereof.
In an exemplified embodiment of the invention, the water soluble polymer
is polyvinyl alcohol.
The method may include employing a cryoprotectant or lyoprotectant in
the emulsion. In one embodiment of the invention, the cryoprotectant or lyoprotectant is
added to the water phase.
The cryoprotectant or lyoprotectant may be selected from the group
consisting of a monosaccharide or a disaccharide, and mixtures thereof.
Preferably, the cryoprotectant or lyoprotectant is one or more of sucrose,
lactose, fructose, mannitol, trehalose, glucose and ribose.
In an exemplified embodiment of the invention, the cryoprotectant or
lyoprotectant is a disaccharide in the form of D-fructose.
The vegetable-based edible oil may be selected from the group consisting
of coconut oil, peanut oil, soya bean oil, olive oil, corn oil, apricot oil, castor oil,
macadamia oil, cottonseed oil, palm oil, rapeseed oil, safflower oil, sunflower oil, almond
oil, hazelnut oil, flaxseed oil, grapeseed oil, sesame oil, rice bran oil, mustard oil and
mixtures of two or more thereof. In exemplified embodiments of the invention, the
vegetable-based edible oil is coconut oil, peanut oil, soya bean oil, olive oil or corn oil.
The method of the invention may thus be characterized by the absence of
organic or inorganic chemically synthesized solvents or oils.
The pharmaceutical active may be water soluble or hydrophilic. The water
soluble or hydrophilic pharmaceutical active, e.g. isoniazid, may be present in the water
phase prior to forming of the emulsion.
The pharmaceutical active may be water-insoluble or hydrophobic. The
water-insoluble or hydrophobic pharmaceutical active, e.g. rifampicin, may be present in
the oil phase prior to forming of the emulsion.
More than one pharmaceutical active may be encapsulated. One or more
pharmaceutical active may be water soluble or hydrophilic. One or more
pharmaceutical active may be water soluble or hydrophilic.
Forming an emulsion from the oil phase and the water phase may include
vigorously emulsifying the oil phase and the water phase together in one embodiment
of the invention, a blender is used to emulsify the oil phase and the water phase
together, with the blender operating at a rotational speed of about 4,000 to about 8,000
rpm.
The water of the water phase may be deionised water.
The emulsion may be lyophilized at a temperature of between about -50 °C
and about -70 °C, preferably between about -55 °C and about -70°C, more preferably
between about -60 °C and about -70 °C, e.g. about -60=.
The emulsion may be lyophilized at a pressure of between about 7.5
Pa(absolute) and about 33.5 Pa(absolute), preferably between about 10.5 Pa(absolute)
and about 25.5 Pa(absoiute), more preferably between about 12.0 Pa(absolute) and
about 20 Pa(absotute), e.g. about .5 Pa(absolute).
The emulsion may be lyophilized for a period of between about 24 hours
and about 120 hours, preferably between about 48 hours and about 1 0 hours, more
preferably between about 72 hours and about 1 0 hours, e.g. about 96 hours.
Lyophilizing the emulsion may include first snap freezing the emulsion by
cooling the emulsion to a freezing temperature below the temperature at which the
emulsion is iyophilszed, typically over a freezing period of at least a few minutes.
The freezing temperature may be between about -60 °C and about -196°C,
preferably between about -100°C and about - 196°C, e.g. about -190°C.
The freezing period may be between about 5 minutes and about 60
minutes, preferably between about 10 minutes and about 40 minutes, more preferably
between about 20 minutes and about 40 minutes, e.g. about 30 minutes.
The particles of the encapsulated pharmaceutical active or the powder
may have an average particle size falling in the nano range. When the particles or
powder has an average particle size falling in the nano range, the average particle size
may be between about 00 nm and about 999 nm, preferably between about 400 nm
and about 950 nm, more preferably between about 400 nm and about 500 nm, e.g.
about 400 nm.
Instead, the particles of the encapsulated pharmaceutical active or the
powder may have an average particle size falling in the micro range. When the
particles of the encapsulated pharmaceutical active or the powder has an average
particle size falling in the micro range, the average particle size may be between about
1 pm and about 5 pm, preferably between about .02 pm and about 1.62 , e.g. about
1.05 pm.
The matrix former and the vegetable-based oil and the pharmaceutical
active may be selected such that the powder of the encapsulated pharmaceutical active
has a dispersity Dm (i.e. based on molecular mass) or polydispersity index from dynamic
light scattering less than 0.95, preferably less than 0.9, more preferably less than 0.6,
even more preferably less than 0.5, still more preferably less than 0.4, e.g. between
about 0.1 and about 0.9, or between about 0.20 and about 0.90, or between about
0.30 and about 0.6, or between about 0.3 and about 0.5, or between about 0.3 and
about 0.4, e.g. about 0.30.
The matrix former and the vegetabie-based oil and the pharmaceutical
active may be selected such that a colloidal dispersion of the particles of the
encapsulated pharmaceutical active or powder in an aqueous medium may has a zeta
potential of at least negative 20.00 mV, preferably between about -20.00 mV and about
-50.00 mV, more preferably between about -23.50 mV and about -46.70 mV, most
preferably between about -32.90 mV and about -46.30 mV, e.g. about -40.00 mV.
The process may show a product yield of at least about 87%, preferably at
least about 95%, more preferably at least about 99%, where the product yield (in
percentage) =
total weight lyophilisate
x 00.
total weight of all ingredients
The lyophilisate may have a drug loading of at least about 70%, preferably
at least about 98%, more preferably at least about 99%, where the drug loading is the
mass percentage of the pharmaceutical active in the lyophilisate.
The invention extends to a lyophilisate and to a powder produced by the
method as hereinbefore described.
The lyophilisate may be as hereinbefore described.
The powder may be as hereinbefore described.
In one embodiment of the invention, the powder produced by the method
as hereinbefore described has a dispersity Dm or polydispersity index from dynamic light
scattering of less than 0.95, with a colloidal dispersion of the powder in an aqueous
medium having a zeta potential of at least negative 20.00 mV.
As set out hereinbefore, the powder produced by the method as
hereinbefore described may have an average particle size falling in the nano range, or
an average particle size falling in the micro range.
The invention also extends to a powder as hereinbefore described, and to
a pharmaceutical composition or formulation which includes a powder as hereinbefore
described.
In particular, the invention extends to a pharmaceutical product which
includes a powder produced by the method of the invention, the powder having a
dispersity m or polydispersity index from dynamic light scattering of less than 0.95, with
a colloidal dispersion of the powder in an aqueous medium having a zeta potential of at
least negative 20.00 m .
Said powder may have an average particle size falling in the nano range,
or an average particle size falling in the micro range.
The powder of the pharmaceutical product may be as hereinbefore
described.
The invention will now be described using the following experiments and
experimental results and by way of the accompanying diagrammatic drawings in which
Figure 1 is an illustration of one embodiment of a direct dispersion emulsification
method or technique in accordance with the invention for producing nano/micro
formulations;
Figure 2 is an illustration showing the (a) drug release profile, (b) transmission
electron microscopic image (magnification = x 25,000), (c) powder XRD diffractogram
and (d) FTIR spectra of an isoniazid-loaded optimized nano/micro formulation prepared
in accordance with the invention;
Figure 3 is an illustration showing the (a) transmission electron microscopic
image (magnification = x 25,000) (b) drug release profile and c) powder XRD
diffractogram of a rifampicin-loaded optimized nano/micro formulation prepared in
accordance with the invention.
Experimental materials
Polyethylene glycol 4000, poly (vinyl alcohol) 87% - 89% hydrofysed,
coconut oil obtained from Cocos nucifera, peanut oil, soybean oil, olive oil, corn oil, ethyl
cellulose - viscosity 22 cP (Ethocel® 22), isoniazid (hydrophilic; Biopharmaceutical
Classifications System (BCS) class borderline l/lll) and rifampicin (hydrophobic; BCS
class I) were purchased from Sigma Chemical Company (St. Louis, United States of
America). D-fructose was obtained from Merck Chemicals (Darmstadt, Germany). A
other reagents utilized were of acceptable analytical grade and were used as obtained.
Preparation of a nano/micro formulation employing a direct
dispersion emulsification technique in accordance with the Box-Behnken
statistical design
15 variant nano/micro formulations were prepared utilizing a direct
dispersion emulsification technique coupled with lyophilisation and dry milling guided
through a 3-level, 3-factor and 3-centre points Box-Behnken quadratic design using
Minitab Statistical Software, Version 16 (Minitab Inc., State College, PA, United States
of America). Three sets of independent variables classified as the water based phase,
oil based phase and homogenization velocity were utilized in preparing each nano/micro
formulation. In more details, the independent variables included:
(i) The water phase made up of different quantities of D-fructose (D-fruc),
polyethylene glycol 4000 (PEG), poly (viny! alcohol) (PVA) 87% - 89%
hydrolysed dispersed in deionised water (DW) represented as 0, 1 and 2
in Tables 1 and 2
(ii) The oil phase was composed of different quantities of ethyl cellulose -
viscosity 22cP (Ethocel 22) (indicated as Eth 22 in Tables 1 and 2)
dispersed in coconut o l (CO) represented as 3, 5 and 7 in Tables 1 and 2,
and
(iii) Homogenization velocity (HV) refers to the speed of the externally
applied mechanical blending force denoted in Tables 1 and 2
Combinations of the above-mentioned independent variables set at
different levels were employed for the manufacturing of each nano/micro formulation so
as to achieve the formation of a stable and effective set of formulations that can be
suitably optimized for intended pharmaceutical application. The lower and upper limits
selected for the independent variables were based on preliminary experiments. Tables
1 and 2 detail the 3-level of independent variables employed and the 3-factor, 3-centre
points Box-Behnken experimental design template made up of 15 variant nano/micro
formulations.
Table 1: Dependent and independent variables employed in the Box Behnken design
Factor Levels
Independent Variables -1 0 + 1
_ water phase (g) 0 ϊ 2
X Oil phase (g) 3 5 7
3 Homogenization velocity (rpm) 4 6 8
Dependent Variables
Zeta Potential (mV)
Y Drug Entrapment Efficiency
Y3 Particle Size (nm)
NOTE: Water phase: 0 - deionised water = 15.0 g, fructose = 0.7 g, polyethylene glycol
4000 = 0.3 g, poly (vinyl alcohol) = 1.5 g; 1 - deionised water 18.0 g, fructose = 1. 1g,
polyethylene glycol 4000 = 0.9 g, poly (vinyl alcohol) = 1.9 g; 2 - deionised water = 2 1g,
fructose = 1.5 g, polyethylene glycol 4000 = 1.5 g, poly (vinyl alcohol) = 2.3 g. Oil
phase: 3 - ethyl cellulose = 0.5 g, coconut oil = 15.0 g, 5 - ethyl cellulose = 1.0 g,
coconut oil = 10.0 g, 7 - ethyl cellulose = 1.5 g, coconut oil - 5 .0 g. Homogenization
velocity: 4 - 4000 rpm, 6 - 6000 rpm and 8 8000 rpm.
Table 2 : Box-Behnken design template for preparing the nano/micro formulations
Formulation DW D-fruc PEG PVA CO (g) Eth 22 HV (rpm)
(g) (g) (g) (g) (g)
1 18.0 1. 1 0.9 1.9 5.0 1.5 4000.0
2 18.0 1. 1 0.9 1.9 15.0 0.5 8000.0
3 2 1 .0 1.5 1.5 2.3 15.0 0.5 6000.0
4 15.0 0.7 0.3 1.5 10.0 1.0 4000.0
5 18.0 1. 1 0.9 1.9 10.0 1.0 6000.0
6 15.0 0.7 0.3 1.5 15.0 0.5 6000.0
7 18.0 1. 1 0.9 1.9 10.0 1.0 6000.0
8 18.0 1. 1 0.9 1.9 15.0 0.5 4000.0
9* 18.0 1. 1 0.9 1.9 0.0 1.0 6000.0
10 15.0 0.7 0.3 1.5 5.0 1.5 6000.0
11 2 1.0 1.5 1.5 2.3 5.0 1.5 6000.0
12 2 1.0 1.5 1.5 2.3 10.0 1.0 4000.0
13 15.0 0.7 0.3 1.5 10.0 1.0 8000.0
14 2 1.0 1.5 1.5 2.3 10.0 1.0 8000.0
15 18.0 1. 1 0.9 1.9 5.0 1.5 8000.0
NOTE: Each formulation contained 1500 mg of isoniazid as model drug
Indicate the design centre points
For each nano/micro formulation, the water phase containing D-fruc, PEG,
PVA and the model drug at this stage, isoniazid, were dispersed in deionised water
(DW) at different levels {Table 2) and the oil phase made up of varying quantities of Eth
22 dispersed in coconut oil (Table 2) were separately prepared under room conditions
(23 °C ± 2°C). Thereafter, the oil phase dispersion was gently added into the water
phase and then immediately subjected to a rigorous, continuous b!ending in the
presence of different magnitudes of an externally applied blending force of a mixer
homogenizer for 5 minutes (Silverson Machines, Inc., Massachusetts, United States of
America) to produce a monophasic emulsion. With D-fructose as a
lyoprotectant/cryoprotectant, each formed emulsion formulation was then snap frozen
by placing it in liquid nitrogen for about 20 minutes. Subsequently, frozen emulsion
samples were then freeze dried/lyophilized (Benchtop Pro Freeze Dryer, VirTis, SP
Scientific, New York, United States of America) at a temperature of -60 ± 2°C and
pressure of 124 ± 2 mtorr (about 16.5 Pa(a)) for 96 hours to produce a solid
lyophilisate/dried cake. For easy handling and testing, the produced lyophilisate was
dry milled using a laboratory scale machine {Kinematica GMBH, Eschbach, Germany)
and stored away in airtight and opaque containers for further testing. Figure 1 illustrates
the direct dispersion emulsification method employed to produce the exemplified
nano/micro formulations of the invention.
Characterization of the 5 experimental design based nano/micro formulations
Determination of particle size, polydispersity index andzeta potential
Measurement of particle size (diameter) and polydispersity index was
based on the principle of dynamic light scattering using the Nano series Zetasizer
equipped with the Zetasizer software, version 6.20 (Malvern Instruments Ltd, Ma!vern,
UK). For all measurements, samples were re-dispersed in distilled water, appropriately
diluted and sonicated (Table-type Supersonic Cleaner KQ1 8 , Nanjing T-Bota Scietech
Instruments and Equipment, Co, Ltd., Jiangsu, China) for 15 minutes at 37°C. All
measurements were performed as three independent replicates with 15 readings per
sample at a measurement angle of 173° and a temperature of 25 °C.
The zeta potential, an indicator of particle surface charge which
determines particle stability in dispersion was computed based on the Smoluchowski
equation using the Nano series Zetasizer equipped with the Zetasizer software, version
6.20 (Malvern Instruments Ltd, Malvern, UK). Each sample was dispersed in an
aqueous media and measurements were carried out in triplicate with 20 runs per each
measurement cycle at 25 C.
Drug loading efficiency
To determine the amount of drug loaded within each nano/micro
formulation variant, 10 mg of each sample was placed in 00 mL phosphate buffer (pH
7.4) and continuously stirred (Five-Position Hot Plate/Stirrer, Model 51450 series, Cole-
Parmer, Illinois, United States of America) for 4 hours to ensure complete dissolution
and release of entrapped drug molecules. The resultant solution was then appropriately
diluted with distilled water and passed through a 0.45 pore size polypropylene
membrane cameo syringe filter (Millipore Corporation, Massachusetts, United States of
America). The actual drug content was analysed using Ultraviolet Spectrophotometry
(PerkinElmer Lambda 35, UV/Vis Spectrometer, Perkin Elmer, Singapore) at a
maximum wavelength of absorption for isoniazid, a = 262 nm. The percentage of
tsoniazid-loaded within each nano/micro formulation was mathematically computed with
reference to the initial quantity that was added into each formulation. All tests were
conducted as three replicate samples.
Evaluation of formulation yield
Produced nano/micro formulations were weighed using a laboratory scale
balance (Kern EG 620-3NM, Kern and Sohn, GmbH, Ba!ingen, Germany). The
percentage yield was calculated utilizing equation 1 below:
In vitro dissolution studies
In vitro dissolution behaviour of the nano/micro formulations was
evaluated employing the dissolution tester (Ewreka GmbH, DT 820 series,
Heusenstamm, Germany). For each test, 500 mg of each nano/micro formulation was
filled into an empty gelatine capsule shell (CapsCanada, Ontario, Canada), placed into
the basket holder attached to the stirring shaft of the dissolution tester (United States
Pharmacopeia Apparatus 1) . The whole contrivance was then immersed into a
dissolution jar containing 500 mL of pre-heated buffer solution (pH .2, pH 6.8 and pH
7.4 separately) at 37 ± 0.5 °C rotating continuously at 100 rpm. 3 mL sample was
collected at pre-determined time-points over 2 hours. Experiments were performed in
triplicate under sink conditions. Collected samples were appropriately diluted, filtered
using a 0.45 pore size polypropylene membrane cameo syringe filter (Millipore
Corporation, Massachusetts, United States of America) and analysed
spectrophotometrical!y (PerkinElmer Lambda 35, UV/Vis Spectrometer, Perkin Elmer,
Singapore) at a ma of 262 nm to determine the amount of tsoniazid contained in every
withdrawn sample. Percentage drug release was computed relative to the total amount
of isoniazid present within the dissolution medium.
Systematic statistical optimization based on the Box-Behnken design template
Experimental outputs from the Box-Behnken quadratic design template
relating independent with dependent variables were subjected to strict settings for
predicting the optimal formulation. A synchronized optimization approach was applied
utilizing the response surface optimizer (Minitab software, Version 16 , Pennsylvania,
United States of America). In this regard, limits were established n order to generate
independent variables that will concurrently influence the response parameters to
generate the desired statistically relevant optimal levels. Consequently, a target was
set for the drug entrapment efficiency, zeta potential while particle size was minimized
(Table 3). A composite desirability vaiue of 0.99 which indicated the robustness and
accuracy of the optimizer was obtained. An estimation of the statistical significance and
dependability of the model was evaluated using the one-way analysis of variance
(ANOVA) with p-values set at 95% confidence level (p 0.05) and correlation coefficient
selected as key indicators (R > 0.80) .
Table 3 : Numerical optimization parameters set for the selected responses
Parameters Optimization Lower Target Upper Desirability
Goal
Zeta Potential (mV) Target 38.00 40.00 42.00 0.99
Drug loading efficiency Target 92.00 96.00 98.00 1.00
( )
Particle size (nm) Minimize 400.00 500.00 700.00 1.00
Preparation and physicochemical characterization of the optimized formulation
Optimized formula
Based on the statistical optimization process described, an optimized
nano/micro formulation was developed. The formula of the optimized formulation is
highlighted in Table 4. The optimized nano/micro formulation was prepared following
the direct dispersion emulsification technique illustrated in Figure 1.
Table 4 : Composition of the statistically optimized formulation
Optimized Formula Components Quantities
Deionised water 18.0 g
D-Fructose 1. 1 g
Polyethylene glycol 0.9 g
Polyvinyl alcohol .9 g
Coconut oil 10.0 g
Ethy! cellulose 1.0 g
Model drug (isoniazid) 1.5 g
Homogenization velocity 4000.0 rpm
Physicochemical characterization of the optimized nano/micro
formulation
Particle size, polydispersity index and zeta potential were determined
based on the method already described hereinbefore. The evaluations of drug loading
efficiency, formulation yield and in vitro dissolution were also performed in accordance
with the methods outlined in hereinbefore. All analyses were conducted three times.
With drug release, the duration of dissolution was increased to 4 hours.
The morphology of the optimized formulation was visualized using
Transmission Electron Microscopy (TEM). About 0.5 mg of test sample was dispersed
in ethanol and spotted on top of a carbon coated copper grid. The ethanol was allowed
to evaporate under atmospheric conditions before the samples were loaded into the
TEM machine viewing stage (JEOL JEM-21 00 LaB6 200 kV Transmission Electron
Microscope, JEOL, Massachusetts, United States of America). Sample viewing under
the TEM machine was facilitated by the Gatan Digital Micrograph Software (Gatan Inc.
California, United States of America).
A qualitative powder x-ray diffraction (XRD) study was performed on the
optimized formulation under room temperature conditions employing an X-ray
diffractometer equipped with the X'Pert PRO data collector software (PANa!ytical Inc.
Massachusetts, United States of America). Approximately 2 - 4 mg of the sample
powder were loaded on standard sample holders and tested. The samples were
continuously scanned between a range of 5.0° and 90.5° with a scan step size of 0.03.
Fourier Transform infra-red (FTiR) spectra of the optimized formulation
were generated on a Perkin Elmer Spectrum 100 Series FTIR Spectrophotometer
coupled with the Spectrum V 6.2.0 software (Beaconsfield, UK). The sample holder
situated on the test stage of the FTIR machine was cleaned with ethanol and allowed to
dry properly. Thereafter, 5 mg of the test sample was placed on the cleaned stage for
structural analysis. Blank background readings were taken prior to sample analysis
which was carried out with wavenumbers ranging from 4000-650 cm 1 , scan time 32
scans and resolution of 4 cm 1. Recorded outputs were computed as an average of
three repeated scans.
Evaluating the effect of loading a hydrophobic drug, rifampicin, onto
the optimized nano/micro formulation
As set out hereinbefore, isoniazid, a hydrophilic molecule, was explored
as the model drug. In order to evaluate the versatility of the direct dispersion
emulsification technique to enable the formulation of hydrophilic or hydrophobic
bioactive molecules, rifampicin, a hydrophobic molecule was incorporated into the
optimized formulation and tested accordingly. Characteristic tests performed included
drug loading efficiency, yield, particle analysis (size, polydispersity index and zeta
potential), morphology and drug release. These tests were conducted based on the
methods described hereinbefore. Outcomes of these measurements were compared
with those generated for the isoniazid-containing formulation. For drug release and
drug loading efficiency analyses for rifampicin, the wavelength of absorption, ma = 338
nm, which is characteristic for this molecule was employed. The quantity of rifampicin
obtainable at this maximum wavelength was mathematically computed. Furthermore,
drug release analysis was performed over a duration of 6 hours to ensure that the
rifampicin molecules are completed released from the within the formulation matrix.
Preparation of the optimized formulation using a variety of
vegetable-based edible oils other than coconut oil
Furthermore, the versatility of the direct dispersion emulsification
technique with regards to varying the oil-phase which is based on the nature of the
vegetable-based oil employed in the manufacture of the stable nano/micro formulation
was also explored. The formulas evaluated were based on the basic composition of the
optimized formulation (Table 4) and are presented in Table 5 below. The produced
formulations were characterized and compared with the earlier-described coconut oilbased
formulation by measuring particle size, polydispersity index, zeta potential,
formulation yield and drug loading efficiency in triplicate.
Table 5 : The statistically optimized formulation prepared with a variety of vegetable
based oils to assess the versatility of the direct dispersion emulsification method
Types of Vegetable Oil Formulation Abbreviation
Coconut oil Opt-CO
Corn oil Opt-CRO
Olive oil Opt-00
Peanut oil Opt-PO
Soybean oil Opt-SO
ther formulation components are exactly the same as reported in Table 4 above. For
each formulation, 10 g of the respective vegetable based oil is used. The model drug
employed in assessing the performance of these formulations was isoniazid.
Stability Testing
Evaluation of stability under different environmental conditions
Stability testing was carried out on both the isoniazid-loaded and
rifampicin-toaded optimized formulations. The different environmental conditions
evaluated were based on three different storage settings:
i) In a stability tester: The samples (500.0 mg ± 2 mg) were placed in an
enclosed glass holder and into a stability tester (Labcon PSIE RH 40
Chamber Standard Incubator, Laboratory Marketing Services, Maraisburg,
South Africa) fixed at settings of 30 C ± 2 C and a relative humidity of
65% ± 3% adapted from the World Health Organization stability testing
scheme for pharmaceutical products containing well established drug
substances.
ii) Under room conditions: Samples (500.0 mg ± 2 mg) were stored in
airtight, opaque glass vials within room conditions (25 °C ± 5°C and
relative humidity 55% ± 5%) for 4 months.
iii) In a refrigerator: Samples (500.0 mg ± 2 mg) were placed in airtight,
opaque glass vials and put into a refrigerator (Sanya Labcool
Pharmaceutical Refrigerator, MPR-720R, Sanyo Electrical Biomedical Co.
Ltd, California, United States of America) at a temperature of 4°C ± 1°C.
Indicators of formulation stability under the different set test conditions
were particle size, polydispersity index, zeta potential, drug content (DC) and physically
examined sample colour evaluated at 0, 1 and 4 months intervals as three replicate
samples.
Assessment of hydrostability in aqueous suspension environment
Hydrostability, which is indicative of the stability of the isoniazid or
rifampicin formulation when exposed to aqueous conditions (e.g. during re-constitution
or re-suspension) was also evaluated. Powdered samples (500 mg) were contained in
airtight, opaque containers and dispersed in sterilized deionised water (50 mL). Each
hydrated test sample was prepared in triplicate and samples were placed under room
conditions (23 °C ± 2°C) as well as concurrently in the refrigerator (4°C ± 2°C). The test
was carried out for 1 days and samples (2 mL) were collected at predetermined time
intervals (0, 1, 5 and 11 days) to assess drug content in triplicate. Prior to the sample
collection and measurement stages, samples were subjected to gentle manual shaking
to ensure uniform re-dispersion.
Experimental results
Quantification of the physical properties of the 15 experimental
design nano/micro formulations
The nano/micro formulations produced based on the Box-Behnken
experimental design template highlighted in Table 2 generated response parameters
which are indicative of their varying measured physical properties. The dependent
variable (responses) studied included particle size or diameter (PS), polydispersity
index (PDI), zeta potential (ZP), drug loading efficiency (DLE), formulation yield (Fy d)
and cumulative drug release over 2 hours in buffer media with variable pH of 1.2
{CDF .2), pH 6.8 (CDR 6.8) and pH 7.4 (CDR ,ra 7.4) and the values are outlined
in Table 6.
The 5 nano/micro experimental design formulations appeared as creamwhite
free flowing, powdery solids. Particle sizes or diameters of the experimental
design formulations spanned over the nano-range of 400.1 0 n ± 8.767 nm and the
micro-range of 624.00 nm ± 19.42 nm with a polydispersity index that was between
0.37 ± 0.01 1 and 0.91 ± 0.1 17 depending on the composition of the respective
nano/micro formulation (Table 6). The zeta potential values outlined in Table 6 showed
that the particulate components of the nano/micro formulations displayed an overall
negatively charged surface characteristics with good stability potential for dispersion as
suspensions as all the formulations excluding F6 (-23.50 V ± 1.05 mV) had zeta
potential values above 30.00 mV. Research has shown that particulate formulations
with zeta potential values greater than (±) 30.00 mV are stable in suspension. Drug
loading within the particulate content of each nano/micro formulation can be described
as quite efficient with values that ranged between 70.04% ± 5.22% and 107.63% ±
3.1 5% (Table 6) and overall average of 88.84%. The direct dispersion emulsion
process can be described as high yielding with regards to the final quantity of the
nano/micro formulations produced with a minimum and maximum yield of 87.43% ±
0.01 % and 95.25% ± 0.1 1% respectively (Table 6 . The formulations displayed diverse
drug release behaviours in the different phosphate buffered media (pH .2, 6.8, 7.4) that
were studied. Drug release behaviour was characterized with a "burst release" for all
formulations followed by consistent release over the 2 hour test duration. Drug release
capacity under each release media condition varied from one formulation to the other
(Table 6). Drug release mimicked a zero order pattern with values of cumulative drug
release at 2 hours ranging from 43.97% ± 2.1 0% 84.06% ± 3.33% (pH 1.2 media),
43.54% ± 0.57% - 83.77% ± 1.01 % (pH 6.8 media) and 33.07% ± 1.47% - 84.49% ±
2.84% (pH 7.4 media).
Physicochemical Characterization of the isoniazid-loaded optimized
nano/micro formulation
The optimized nano/micro formulation was prepared in accordance with
the formula systematically derived through the statistical optimization procedure (Table
4). The optimized formulation had an average particle size of 428.14 nm ± 25.54 nm,
zeta potential of -38.68 mV ± 3.21 mV, polydispersity index of 0.38 ± 0.04, drug loading
efficiency of 97.13% ± 1.05% and process yield of 98.02% ± 0.01 %. The zeta potential,
drug loading efficiency and particle size were initially selected as pointers for the
statistical optimization of the nano/micro formulation (Table 3). The obtained
experimental values were closely related to the statistically predicted values (see Table
7 below). This outcome revealed that the high performance quadratic experimental
design template selected for the formulation optimization was not only suitable for its
intended application but was also accurate, stable and robust.
Table 6: Numerical values of the response parameters generated for the 5 experimental design Nano/micro formulations
Nano/W!icro PS (nm) p ZP (mV) DLE {%) Fyie|d {%W/W) CD .2 CD _¾rs6.8 C 2hrs7A
Formulations (%) { ) (%)
1 525.1 0 0.46 -38.1 0 83.67 94.21 84.06 79.55 77.42
2 439.40 0.38 -34.20 0 1.30 92.88 45.26 43.54 33.07
3 901 .60 0.69 -32.90 97.92 92.92 79.46 80.87 62.87
4 400.1 0 0.37 -35.20 85.04 89.85 75.94 83.77 63.70
5 498.24 0.43 -42.41 92.05 93.90 70.92 54.08 47.34
6 1022.20 0.79 -23.50 9 1.89 88.34 78.49 80.85 62.56
7 499.04 0.44 -40.61 93.45 94.82 7 1.33 54.96 49.38
8 492.40 0.43 -40.71 8 1.71 90.15 57.63 7 1.89 48.77
9 497.98 0.44 -39.96 93.49 94.85 7 1.33 55.91 49.80
10 484.01 0.53 -38.90 70.04 95.25 43.97 68.32 67.44
11 578.06 0.45 -40.80 80.42 93.33 62.60 6 1.31 84.49
12 1624.00 0.38 -41 .12 94.12 87.43 67.88 60.18 49.36
13 066.05 0.91 -46.70 79.97 92.46 66.94 68.10 65.13
14 949.90 0.75 -45.51 94.42 94.45 69.72 56.78 48.25
15 946.1 0.74 -46.30 68.89 9 1.55 53.93 69.19 60.61
Particle Size (Standard Deviation < 2 3 5 nm in all cases), Polydispersity Index (Standard Deviation 0. 16 in all cases), Zeta Potentia
(Standard Deviation 10.60 mV in all cases), d Drug Loading Efficiency (Standard Deviation 16.88% in all cases), Formulation Yield (Standar
Deviation 0.54% in all cases), ! Cumulative Drug Release (pH 1.2) (Standard Deviation 6.83% in all cases), Cumulative Drug Release (pH 6.8
(Standard Deviation 9.72% in all cases),* Cumulative Drug Release (pH 7.4) (Standard Deviation 7. 14% in all cases) (N = 3 in all cases)
Table 7 : Comparison of predicted versus experimental values of the optimization
parameters
Optimization Parameters Predicted Values Experimental values
Zeta Potential (mV) -40.00 -38.68
Drug loading efficiency (%) 96.00 97.1 3
Particle size (nm) 500.00 428.14
In vitro isoniazid release from the optimized nano/micro formulation was
characterized with an initial burst from 5 minutes ( 1 0.51 %) which increased at a
consistent rate up until 40 minutes, followed a slower significant increase in percentage
release up to 60 minutes after which a plateau was reached where drug release
remained more or less consistent up to the 240 minutes time point when about a 100%
release was achieved. To further analyse the in vitro release profile, a variety of kinetic
models were utilized for the model fitting process using free open source software
(KinetDS, version 3.0). The selection of the choice model which fitted the release data
the best was based on the correlation coefficient (R ) with values closest to 1. On this
basis, the in vitro drug release kinetics of the isoniazid-loaded optimized nano/micro
formulation was best expressed by the Michaelis-Menten (R2 = .00) and Korsmeyer
Peppas (R = 0.99) equation because both fits displayed the best linearity. These
mathematical models indicate that drug release is mono-dimensional from the polymerbased
platform. Besides, the diffusion exponent (n) displays values greater than 0.89
with the Michaelis-Menten model, n = 1.00 and the Korsmeyer Peppas model, n = 1.01 .
The obtained n values signify that the release trend follows a super case li transport
mechanism. Consequently, it can be inferred that isoniazid release from the optimized
nano/micro formulation is controlled by stress induced relaxation and state transition
within the polymeric chain resulting in swelling followed by erosion of polymeric chain
and irregular diffusion of drug molecules.
The TEM image of the isoniazid-loaded optimized nano/micro formulation
is shown in Figure 2 (b). The particulate composition of the nano/micro formulation can
be described as spherical in geometry (highlighted by the red arrows in Figure 2 (b)).
The powder XRD diffractogram of the formulation is shown in Figure 2 (c). The
characteristic broad peaks observed from the generated XRD diffractogram showed that
the isoniazid-loaded nano/micro formulation can be referred to as semi-crystalline in
nature. The observed diffraction pattern (Figure 2 (c)) showing a number of different
blunt peaks further indicate that the formation of the nano/micro formulation is not
chemical in nature but due to a physical interaction between the individual component
compounds. Furthermore, the nature of the produced diffractogram peaks confirms the
presence of polymeric components within the formulation. The semi-crystalline nature
of this formulation can explain why its dissolution rate is relatively quick based on its
drug release behaviour which is characterised by the initially elicited burst effect
followed by almost a 100% release in about 2 hours (Figure 2(a)).
The peaks depicting characteristic vibrational frequencies of isoniazid in
its pure state and loaded in the formulation were compared. For pure isoniazid, bond
vibrational peaks recorded included C-C-C-C and C-N-C-C torsion (653 cm 1) , NH2 rock
(677 cm 1 ) C-C-C and C-C-H out of plane bending (746 cm" 1) , C-C-H out of plane
bending (850 cm 1; 1022 cm 1 ) , C-N-C and C-C-C in plane bending ( 1063 cm" 1) , C-C-H
in plane bending ( 1210 cm 1), 0=C-N and C-C-H in plane bending ( 1330 cm 1) , C-N-H in
plane bending (141 cm 1 ) , C-C stretching ( 77 cm 1) , C=0 stretching ( 1 558 cm 1) , NH
scissoring ( 1632 cm 1 ) , aromatic C-H stretching (3052 cm 1 ) and N-H stretch (3307 cm
) . The vibrational peaks documented for isoniazid in the nano/micro formulation were
C-C-C-C and C-N-C-C torsion (665 cm 1 ), NH rock (677 cm 1 ) C-C-C and C-C-H out of
plane bending (757 cm 1 ), C-C-H out of plane bending (850 cm 1 ; 1023 cm" 1 ) , C-N-C and
C-C-C in plane bending (1062 cm" 1) , C-C-H in plane bending (121 2 cm" 1 ) , 0=C-N and
C-C-H in plane bending ( 1 333 c ) , C-N-H in plane bending (1408 cm ) , C-C
stretching (1471 cm 1 ) , C=0 stretching ( 1552 cm" 1 ) , NH2 scissoring (1635 cm 1 ) ,
aromatic C-H stretching (3054 cm 1) and N-H stretch (3308 cm 1 ) . Comparing the two
sets of vibrational frequency readings for isoniazid in the pure and formulation states, a
closely related trend in the magnitudes of the vibrational frequencies is notable. These
similarities indicate that the nano/micro formulation is a physical mixture of active drug
plus excipients and not a chemtcal, irreversible or destructive interaction among the
component compounds.
Characterization of the rifampicin-loaded optimized nano/micro
formulation
The rifarnptcin-loaded formulation had a drug loading efficiency of 98.83 ±
1.23%, a process yield of 98.87 ± 0.02% with an average particle size of 429.37 ± 28.65
nm, zeta potential of -36.10 ± 2.89 V and polydispersity index of 0.37 ± 0.03.
Particulate geometry of the rifampicin-loaded formulation can be described as spherical
as illustrated with the arrows in Figure 3 (a) from the TEM micrograph.
In vitro drug release (Figure 3 (b)) followed a similar pattern with that of
the isoniazid-loaded formulation but with an extended duration. Drug release behaviour
of the rifampicin-loaded formulation was also characterized by a burst release which
was initiated at 5 minutes (3.93%) and increased steadily until 120 minutes (94.46%)
after which drug release was more or less consistent up to 360 minutes when about a
100% release was achieved. In a nutshell, the rifampicin release pattern from the
optimized nano/micro formulation followed a similar trend when compared with the
isoniazid-loaded formulation. However, rifampicin release rate was slower than that of
isoniazid and this is attributable to difference in the aqueous solubility of both model
drugs. Additionally, the release profile of the rifampicin-loaded formulation was
subjected to model fitting procedures employing the free open source software
(KinetDS, version 3.0). The best fit was based on the value of the correlation coefficient
(R2) with values closest to . With reference to this, the in vitro release mechanism of
this formulation was best explained by the Michaelis-Menten (R2 = 1.00) and Korsmeyer
Peppas (R 0.97) based on the R2 values. This indicates that drug release from the
formulation is unidimensional. Also the n {diffusion exponent) values > 0.89 (Michaelis-
Menten model, n 0.99 and the Korsmeyer Peppas model, n = 1.20) implies that the
drug release mechanisms of the rifampicin-loaded formulation is also regulated by
stress induced relaxation and polymeric chain transition which leads to matrix swelling,
erosion and diffusion modulation (super case I I transport). Overall, the optimized
nano/micro formulation is a robust and flexible carrier system that can effectively
regulate the release of either hydrophilic or hydrophobic drugs/bioactive moieties.
The nature of the powder XRD diffractogram (Figure 3(c)) was majorly
characterised by blunt, not well defined peaks {amorphous domains) in terms of
sharpness and a few distinct peaks (crystalline domains) showing that the formulation is
semi-crystaliine and contains multiple compounds. This indicates that no irreversible
chemical transitions occurred during the production of the rifampicin-loaded nano/micro
formulation. This also confirms the nature of the release profile which is characterised
as a relatively quick drug release rate.
Testing of optimized nano/micro formulation prepared with alternate
vegetable-based oil phase other than coconut oil
The produced drug nano/micro formulation variants based on alternate
vegetable oils (corn, olive, peanut and soybean oils) as oil phases had a high process
yield (95.56% - 99.81%), drug loading capacity (93.83%-98.77%), nano-range
particulate dimensions of 277.96 n - 7 18.58 nm, polydisperstty index ranging between
0.32 0.47 and zeta potential of 29.85 V - 34.84 mV (Table 8). These values are
related to the values obtained for the optimized formulation with oil phase solely made
up of coconut oi .
Table 8 : Physicochemical characteristics of the optimized nano/micro formulation
prepared using alternative vegetable-based edible oil
This represents the original/lead optimized nano/micro formulation
Formulation Stability Evaluation
Varying environmental condition stability testing
Outcomes of the performed stability test measured by the values of the
stability indicators (polydispersity index, zeta potential, particle size, drug content and
colour change) are presented in Tables 9 (a), 9 (b) and 9 (c). After 4 months of storage
of the drug loaded optimized formulations, slight, statistically insignificant (p>0.05)
changes in the values of the stability indicators were noticed for all three testing
conditions (stability tester - 30 C ± 2°C and 65% ± 3%, room - 25 ± 5°C and 55% ±
5% and refrigerator - 4°C ± 1°C). However a slight colour change from pure white to
cream-white was observed for the isoniaztd-loaded formulation stored under room and
stability tester conditions (Tables 9 (a) and (b)). Nevertheless, both model drugs were
well preserved within the formulation under all storage conditions. Considering all the
stability indicators measured under all the respective storage condition, storage of this
formulation under refrigeration conditions (4 C ± 1°C) (Table 9 (c)) appears to be the
most suitable storage conditions, as no discoloration was observed for all the
formulations and changes in physical properties were slight and statistically insignificant
(p>0.05).
Table (a): Values of stability indicators at the different time-points for stability
experiments conducted within the stability tester environment
Nano/Micro Test Time Stability indicators
Formulation Points
(months)
PDI ZP (mV) PS DC (%) Colour
(nm) changes
0 0.38 -38.68 428.14 97.1 3 None
Isoniazid 1 0.41 -35.59 429.50 97.07 None
4 0.39 -33.60 403.55 96.76 Slight
Rifampicin 0 0.37 -36.1 0 429.37 98.83 None
1 0.36 -35.80 402.20 98.32 None
4 0.35 -35.1 0 4 10.71 98.70 None
a Particle Size (Standard Deviation 14.61 nm in all cases ~Polydispersity Index
(Standard Deviation < 0.02 in all cases), Zeta Potential (Standard Deviation 3.21 mV
in all cases), d Drug Content (Standard Deviation 1.23% in all cases) (N = 3 in all
cases).
Table 9 (b): Values of stability indicators at the different time-points for stabiiity
experiments conducted under room conditions
Nano/Micro Test Time Stability indicators
Formulation Points
(months)
PDIa PS DC^(%) Colour
(nm) changes
0 0.38 -38.68 428.14 97.1 3 None
Isoniazid 1 0.44 -39.7 439.9 96.95 None
4 0.46 -37.1 4 19.78 97.05 Slight
Rifampicin 0 0.37 -36.1 0 429.37 98.83 None
1 0.39 -34.30 409.40 98.04 None
4 0.36 -33.96 403.70 97.99 None
Particle Size (Standard Deviation 10. 11 nm in all cases), Polydispersity Index
(Standard Deviation 0.04 in all cases), Zeta Potential (Standard Deviation 1.85 mV
in all cases), d Drug Content (Standard Deviation 2.21% in all cases) (N = 3 in all
cases).
Table 9 (c): Values of stability indicators at the different time-points for stability
experiments conducted in the refrigerator environment
Nano/Micro Test Time Stability indicators
Formulation Points
(months)
_ _ p zP m ) PS £ DC %) Colour
(nm) changes
0 0.38 -38.68 428.14 97.1 3 None
Isoniazid 1 0.38 -36.65 426.1 0 96.95 None
4 0.35 -35.91 431 .30 97.02 None
Rifampicin 0 0.37 -36.1 0 429.37 98.83 None
1 0.37 -34.21 418.85 98.88 None
4 0.36 -34.98 4 11.99 99.01 None
a Particle Size
(Standard Deviation 0.01 in all cases), Zeta Potential (Standard Deviation < 1,43 mV
in all cases), Drug Content (Standard Deviation 1.48% in all cases) (N = 3 in all
cases)
Hydrostability evaluation
The isoniazid and rifampicin loaded formulations were stable in the
aqueous medium over the specified test period ( 1 1 days) and test condition (ambient
and refrigerator) based on the values of the measured stability indicators outlined in
Table 10. The drug content did not change significantly showing that the encapsulated
model drugs were stable within the hydrated nano/micro matrix. Furthermore, this
outcome reveals the usefulness of this nano/micro formulation in compounding
pharmaceutical suspensions for re-constitution purposes.
Table 0: Values of stability indicators measured at different time-points visualizing the impact of an aqueous environment on the stability
of the optimized formulation stored under ambient or refrigerated conditions
Test Conditions Nano/Micro Test Time Point Hydrostability Indicators
Formulation (days)
DC {%) ZP (mV) PS (nm) P Discoioration
0 97.1 3 ± 1.23 -38.68 ± 1.43 428. 14 ± 12.62 0.38 ± 0.01 No e
1 97. 1 ± 0.78 -38.01 ±0.88 433.45 ± 8.42 0.36 ± 0.06 None
isoniazid 5 96.66 ± 0.56 -36.55 ± 3.25 456.10 ± 6.53 0.37 ± 0.04 None
ROOM/AMBIENT 11 96.98 ± 1.99 -35.91 ± 0.58 481 .80 ± 1.32 0.39 ± 0.01 None
0 98.83 ± 2.05 -36.10 ± 1.26 429.37 ± 12.1 1 0.37 ± 0.02 None
1 97.1 6 ± 1.88 -37.22 ± 2.77 396.09 ± 8.1 0.38 ± 0.04 No e
Rifampicin 5 96.99 + 2.13 -36.85 ± 0.99 4 16.14 ± 4.76 0.36 ± 0.01 None
11 97.94 ± .87 -37.09 ± 2.14 426.31 ± 5.63 0.37 ± 0.03 None
0 97. 13 + 1.23 -37.02 ± 1.76 428.14 ± 12.62 0.38 ± 0.01 None
1 95.29 .02 -37.91 ± 0.97 423.45 ± 4.01 0.38 ± 0.02 None
Isoniazid 5 94.81 ±0.69 -37.79 ± 3.56 448.96 ± 4.27 0.37 ± 0.01 None
REFRIGERATOR 11 94. 19 ± 2.01 -37.53 ± 1.22 472.20 ± 4.44 0.37 ± 0.01 None
0 98.83 ± 2.05 -36. 10 ± 1.26 429.37 ± 12.1 1 0.37 ± 0.02 None
1 98.99 ± 1.02 -37.55 ± 2.26 398.92 ± 7.35 0.38 ± 0.05 None
Rifampicin 5 98.54 ± 2.55 -36.89 ± 3.01 408.09 ± 4.52 0.37 ± 0.01 None
11 98.97 ± 0.89 -37.02 ± 1.76 426.30 ± 1.23 0.38 ± 0.03 None
The method of the invention advantageously can provide nano/microsized
environmentally stable pharmaceutical formulations in an oil-polymer carrier
system, combining an oieo-polymeric technique with lyophilisation and optionally a dry
milling process. The method of the invention, as illustrated, uses an oleo-polymeric
technique which is conservative, simple and convenient, involving a direct dispersion
emulsification approach which makes use of less stringent room conditions, in particular
room temperature for mixing/emulsification, and use biologically compatible solvent
systems such as water and edible vegetable oils that are free of toxic organic solvents
and biocompatible and biodegradable United States Food and Drug Administration
(USFDA), Generally Recognized as Safe (GRAS) polymeric and non-polymeric
additives. The pharmaceutical formulations are formed by physical, non-destructive
interactions between pharmaceutical active and polymeric and non-polymeric
excipients. The ingredients are agitated in the presence of a carefully adjusted
externally applied blending force. Advantageously, the method of the invention, as
illustrated, produces stable nano/micro configured formulations with attractive qualities,
which are useful for drug delivery applications, such as very high loading efficiency and
product yield, controlled release and good transmembrane permeation characteristics,
fiexibfe characteristics in the sense that it can load either hydrophobic or hydrophilic
drug molecules as well as accommodate the use of different encapsulating materials
such as the edible vegetable oils and polymeric and non-polymeric additives. The
formulations produced by the method of the invention, as illustrated, undergo stress
induced polymeric chain relaxation/transition followed by matrix swelling, erosion and
diffusion to release drug molecules. The pharmaceutical formulations prepared by the
method of the invention, as illustrated, have the capability of regulating the influx of
body fluids into their matrices and therefore to release the encapsulated pharmaceutical
active at different administrations sites or through different administration routes, such
as via the oral route or via the transmucosa! route.
Generally, conventional methods of micro/nano encapsulation in contrast
are cumbersome, labour intensive, requires strict conditions relating to temperature and
pressure and the like, are performed in the presence of toxic solvents, are difficult to
scale-up, are expert-dependent and low-yielding, have a low encapsulation efficiency
and are non-flexible in terms of loading hydrophilic and hydrophobic drugs. Overall, the
method of the invention, as illustrated, can provide pharmaceutical formulations that can
be applied orally (e.g. in the form of capsules or suspensions) and/or are oromucosal
dispersible (e.g. in the form of films, gels or wafers).
Claims
1. A method for encapsulating a pharmaceutical active, the method including
dispersing a matrix former into at least one of a water phase and an oil phase,
with the pharmaceutical active being present in at least one of the oil phase and the
water phase and with the oil phase comprising a vegetabie-based edible oil;
forming an emulsion from the oil phase and the water phase; and
lyophi!izing the emulsion to form a solid lyophilisate.
2. The method of claim 1, which includes comminuting the solid lyophilisate
to provide particles or a powder of the encapsulated pharmaceutical active.
3 . The method of claim 1 or claim 2 , in which a hydrophilic matrix former is
dispersed into the water phase and a hydrophobic matrix former is dispersed into the oil
phase.
4. The method of claim 3, in which the hydrophilic matrix former is a
polymeric matrix former, the hydrophilic polymeric matrix former being selected from the
group consisting of a polyethylene glycol, polyacrylic acid, polyether, polyacrylamide,
polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, polyoxazoline,
polymethacryiate, polyethy!enimine, polyphosphate based polymer and mixtures of two
or more thereof, and/or in which the hydrophobic matrix former is selected from the
group consisting of a cellulose ether, a poly (lactide-co-g!ycolide) based polymer, and
mixtures of two or more thereof.
5. The method of any of claims 1 to 4, which includes employing a water
soluble polymer as a surfactant or emulsifying agent for the forming of the emulsion
from the oil phase and the water phase.
6. The method of any of claims 1 to 5, which includes employing a
cryoprotectant or lyoprotectant in the emulsion, the cryoprotectant or lyoprotectant being
selected from the group consisting of a monosaccharide or a disaccharide, and mixtures
thereof.
7. The method of any of claims 1 to 6, in which the vegetable-based edible
oil is selected from the group consisting of coconut oil, peanut oil, soya bean oil, olive
oil, corn oil, apricot oil, castor oil, macadamia oil, cottonseed oil, palm oil, rapeseed oil,
safflower oil, sunflower oil, almond oil, hazelnut oil, flaxseed oil, grapeseed oil, sesame
oil, rice bran oil, mustard oil and mixtures of two or more thereof.
8. The method of any of claims 1 to 7, in which the emulsion is lyophilized at
a temperature of between -50 °C and -70 °C and/or in which the emulsion is lyophilized
at a pressure of between 7.5 Pa(absolute) and 33.5 Pa(absolute) and/or in which the
emulsion is lyophilized for a period of between 24 hours and 0 hours.
9. The method of any of claims 1 to 8, in which lyophilizing emulsion includes
first snap freezing the emulsion by cooling the emulsion to a freezing temperature below
the temperature at which the emulsion is lyophilized, the freezing temperature being
between -60 °C and -196 °C.
10. The method of any of claims 1 to 9, in which the pharmaceutical active is
water soluble or hydrophilic and is present in the water phase prior to forming of the
emulsion, and/or in which the pharmaceutical active is water-insoluble or hydrophobic
and is present in the oil phase prior to forming of the emulsion.
11. The method of claim 2 , in which the matrix former and the vegetablebased
oil and the pharmaceutical active are selected such that the powder of the
encapsulated pharmaceutical active has a dispersity Dm or polydispersity index from
dynamic light scattering of less than 0.95.
12. The method of claim 2 or claim 11, in which the matrix former and the
vegetable-based oil and the pharmaceutical active are selected such that a colloidal
dispersion of the powder of the encapsulated pharmaceutical active in an aqueous
medium has a zeta potential of at least negative 20.00 mV.
13. A powder produced by the method of claim 2, the powder having a
dispersity Dm or polydispersity index from dynamic light scattering of less than 0.95, with
a colloidal dispersion of the powder in an aqueous medium having a zeta potential of at
least negative 20.00 mV.
14. The powder of claim 13, which has an average particle size falling in the
nano range, or which has an average particle size failing in the micro range.
15. A pharmaceutical product which includes the powder of claim 13 or claim
14.