FIELD OF THE INVENTION
The invention relates to nanoparticle carriers for oral administration of medically active
compounds and/or other compounds.
BACKGROUND TO THE INVENTION
The spray-drying technique has seen wide application in the preparation of
pharmaceutical powders, mostly for pulmonary drug delivery, with specific
characteristics such as particle size, density and shape. It is a well-established method for
producing solid powder by atomising suspensions or solutions into droplets followed by a
drying process in flowing hot air.
Although most often considered as a dehydration process, spray-drying can also be used
as an encapsulation method where active substances are entrapped in a polymeric matrix
or shell. It is reported that several colloidal systems such as emulsions or liposomes were
successfully spray dried with preservation of their structure using drying-aid agents,
particularly sugars such as lactose, sorbitol and trehalose.
One of the merits of the spray-drying technique is that it is a cost effective and quick
drying process applicable to a broad range of pharmaceutical products and leading to the
production of a free flowing powder, characterized by very low water content, preventing
therefore the degradation of the active. This is meaningful for the development of longterm
stable carriers, mostly when these carriers are in the range of nano scale, designed
specifically for the delivery of active compounds at the site of interest.
Recently, it has been shown that the spray drying technique can produce nano scale solid
particles and solid lipid nanoparticles loaded with active agents to be used as delivery
systems for pulmonary airways. It is worthwhile to note that in most cases where this
technique was applied to produce solid nanoparticles, it was, in fact, a drying process of
nanocapsules obtained by other techniques. Thereafter the suspension of the
nanoparticles was subjected to spray drying. This resulted often in the production of
3
particles with very broad size range from nano to micron size, despite the presence of
disaccharides as drying excipients in the formulation.
Recently, it was reported the spray drying of a liquid colloidal system in the drug delivery
field, where a single emulsion (water-in-oil emulsion) containing DNA encapsulated in
poly(lactic-co-glycolic acid (PLGA), was successfully spray dried. Another report was
made on spray drying of a double emulsion (oil-in-water-in-oil or O/W/O), in the
presence of lactose, aiming to preserve orange oil and in both cases the particles
produced were in the micron size range.
A need has been identified for spherical nanoparticles having a narrow size distribution
range, typically from 180 to 250 nm. Ideally such particles should have a substantially
smooth surface and be free flowing.
STATEMENT OF THE INVENTION:
Accordingly the present invention provides a process for production of nanoparticle
carriers for drug delivery, said nanoparticles being produced by:
- preparing a double emulsion of water-oil-water including one or more polymer
which forms the basis of the nanoparticle carrier;
- blending the drug to be delivered into one of the emulsion phases;
- doping either the oil-phase or the outer-water phase with a carbohydrate;
- doping either the oil-phase or the water phase with a surfactant; and
spray drying the emulsion to form nanoparticles of a narrow particle size distribution of
100 nm to 1000 nm.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Figure 1: shows SEM micrographs of INH-loaded PLGA nanoparticles spray dried: A.
using DCM; B. using EA; C. DCM+10% (w/v) lactose and D. EA+10% (w/v) lactose.
4
Figure 2: shows size and zeta potential vs. PVA concentration for formulations where
lactose was used without surfynol 104 PG-50TM and PEG.
Figure 3: shows SEM photos of spray dried INH-loaded PLGA nanoparticles. A.
Formulation without stearic acid and PEG and B. formulation with stearic acid and
PEG(Measuring bars represent both 200nm).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is in relation to a process for production of nanoparticle carriers for
drug delivery, said nanoparticles being produced by:
- preparing a double emulsion of water-oil-water including one or more polymer
which forms the basis of the nanoparticle carrier;
- blending the drug to be delivered into one of the emulsion phases;
- doping either the oil-phase or the outer-water phase with a carbohydrate;
- doping either the oil-phase or the water phase with a surfactant; and
- spray drying the emulsion to form nanoparticles of a narrow particle size
distribution of 100 nm to 1000 nm.
In another embodiment of the present invention, the nanoparticles thus produced are
multifunctional nanoparticles.
In still another embodiment of the present invention, the carbohydrate is a saccharide.
In yet another embodiment of the present invention, the saccharide is a disaccharide.
In yet another embodiment of the present invention, the disaccharide is selected from the
group including lactose, maltose, isomaltose, mannobiose, trehalose, and cellobiose.
In yet another embodiment of the present invention, the saccharide is combined with a
cationic biodegradable muco-adhesive polysaccharide.
In yet another embodiment of the present invention, the polysaccharide is chitosan and/or
derivatives thereof.
5
In yet another embodiment of the present invention, the surfactant is a non-ionic
surfactant.
In yet another embodiment of the present invention, the surfactant is based on acetylenic
diol chemistry.
In yet another embodiment of the present invention, the surfactant is a polymeric nonionic
surfactant.
In yet another embodiment of the present invention, the polymeric non-ionic surfactant in
the water-phase is PVA.
In yet another embodiment of the present invention, there is a polymer in the oil-phase of
the emulsion.
In yet another embodiment of the present invention, the polymer in the oil-phase is
PLGA (poly(lactic-co-glycolic acid)).
In yet another embodiment of the present invention, polymers are present in both the oilphase
and the water-phase.
In yet another embodiment of the present invention, the drug is added to the oil-phase.
In yet another embodiment of the present invention, the drug is a hydrophilic drug which
is added to the internal water-phase.
In yet another embodiment of the present invention, the drug is hydrophobic.
In yet another embodiment of the present invention, the drug is selected from Rifampicin,
Isoniazid, Ethambutol, or Pyrazynamide.
In yet another embodiment of the present invention, the outer water-phase of the
emulsion includes polyethylene glycol (PEG).
In yet another embodiment of the present invention, the oil-phase includes stearic acid.
In yet another embodiment of the present invention, the nanoparticles thus formed are
substantially spherical.
6
In yet another embodiment of the present invention, the particle size distribution of the
nanoparticles is from 180 nm to 250 nm diameter.
The invention provides a process for the production of nanoparticle carriers for drug
delivery, said nanoparticles being produced by:
- preparing a double emulsion of water-oil-water including one or more polymer which
forms the basis of the nanoparticle carrier;
- blending the drug to be delivered into one or more of the emulsion phases;
- doping either the oil-phase or the outer-water phase with a carbohydrate; and - spray
drying the emulsion to form nanoparticles of a narrow particle size distribution of 100 nm
to 1000 nm.
The nanoparticles thus produced may be multifunctional nanoparticles.
The carbohydrate may be a saccharide.
The saccharide may be a disaccharide.
The disaccharide may be lactose, maltose, isomaltose, mannobiose, trehalose, cellobiose,
or the like.
The saccharide may be combined with a cationic biodegradable muco-adhesive
polysaccharide.
The polysaccharide may be chitosan or derivatives thereof.
The oil-phase of the emulsion may be doped with a surfactant. The water-phase of the
emulsion may be doped with surfactant.
The surfactant may be a nonionic surfactant.
The surfactant may be based on acetylenic diol chemistry.
7
The surfactant may be a polymeric nonionic surfactant.
The polymeric nonionic surfactant in the water-phase may be polyvinyl alcohol (PVA),
partially hydrolysed.
The polymer may be in the oil-phase of the emulsion.
The polymer in the oil-phase may be PLGA (poly(lactic-co-glycolic acid)).
Both oil-phase and water-phase polymers may be present.
The drug may be added to the oil-phase.
The drug may be a hydrophilic drug which is added to the internal water-phase.
The drug may be hydrophobic and may optionally be added to the oil phase.
The drug may be Rifampicin, Isoniazid, Ethambutol, or Pyrazynamide.
The outer water-phase of the emulsion may include polyethylene glycol (PEG).
The oil-phase may include stearic acid.
The nanoparticles thus formed may be substantially spherical.
The particle size distribution of the nanoparticles may be from 180 nm to 250 nm
diameter.
The description of embodiments which follows should be interpreted broadly and not to
limit the scope of the invention.
SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION
1. Object of Experiment
8
For this experiment, anti-tuberculosis antibiotics including isoniazid (INH) ethambutol
(ETH), pyrazynamide (PZA) and Rifampicin have been successfully loaded in polymeric
core-shell nanoparticles of poly DL, lactic-co-glycolic acid (PLGA50:50), a
biodegradable and biocompatible polymer, extensively used as a carrier. Submicron solid
particles of PLGA incorporating INH (or Eth or PZA or RIF) have been obtained by
spray drying straightforward a typical double emulsion water-in-oil-in-water (W/Oλ/V).
In the formulation, chitosan, a cationic biodegradable muco-adhesive polysaccharide, was
employed as absorption enhancer while lactose monohydrate was used as spray dryingaid.
PVA was considered as the main stabiliser component of the double emulsion, while
PEG was incorporated to increase the bio-circulation of the carrier.
Surfynol 104 PG-50 ™, as a co-surfactant, played a big role in decreasing the particle
size towards the nanosize range while significantly narrowing the size distribution.
2. Materials and Methods
2.1 Materials
The frontline anti-tuberculosis drugs were purchased from Sigma. Poly, DL, Lactic-co-
Glycolic Acid, (PLGA) 50:50 (Mw: 45000-75000) and chitosan low Mw, 85% deacetylated,
were both supplied by Sigma. Polyvinyl alcohol (PVA) (Mw: 13000-23000
and partially hydrolysed (87-89%) was also obtained from Sigma. Stearic acid supplied
by Merck, Surfynol 104 PG-50 ™, a Gemini diol type surfactant, was supplied by Air
Products. Polyethylene glycol (PEG) (Mw 9000) was purchased from BASF Chemicals.
Lactose monohydrate supplied by Merck, was used as an excipient.
Dichloromethane, ethyl acetate and acetonitrile, analytical and HPLC grades were also
supplied by Merck.
2.2 Methods
2.2.1 Formulation
9
The preparation of nanoparticles was achieved by the method based on the interfacial
polymer precipitation from a double emulsion W/Oλ/V subsequent to the evaporation of
the organic solvent. In this invention, the step of solvent evaporation and drying was
combined in one step by applying the spray drying technique.
Briefly, 50mg of INH was dissolved in a 2ml of phosphate buffer solution (pH7.4), which
was added to a solution of 100mg of PLGA (50:50) dissolved in 8ml of the organic
solvent (DCM or ethyl acetate). An optional 2ml of 0.2%(w/v) of stearic acid can also be
dissolved in the same solvent (DCM or Ethyl acetate). A drop of Surfynol 104 PG-50 ™
was intentionally added either to the PLGA oil phase or to the external aqueous phase
containing PVA.
The mixture was subject to emulsification using the high speed homogeniser (Silverson
L4R) at 5000 rpm for 3min to produce W/O emulsion. This first emulsion obtained was
then immediately poured into an aqueous phase volume of a known concentration of
PVA (1 or 2% w/v), PEG 0.5% w/v, chitosan and lactose aqueous solution in a defined
volume ratio, and emulsified to form the double emulsion W/C7W again by means of the
high speed homogenizer (Silverson L4R) at 8000rpm for 5min. The final emulsion
obtained was directly fed through a spray dryer to produce nanoparticles using the
conditions specified in Table 1.
Spray drying
A Bϋchi mini spray dryer model B-290 (Bϋchi Lab, Switzerland) with a standard nozzle
(0.7 mm diameter) was used to produce the dry powders of the various formulations. The
conditions used are compiled in Table 1:
Table 1: Spray-drying process condition of B-290 Bϋchi Mini Spray Drier
Condition Parameter
Atomizing air volumetric flow rate 800 NL/h
Feeding rate 1.0 mL/min
10
Aspirator rate 100%
Inlet (outlet) temperature 90 -1100C (53-63°C)
Pressure for atomisation 6-7 bars
The spray dryer was provided with a high performance cyclone, designed to get an
excellent recovery of the material in the receiver vessel and reduce the adhesion of the
product on the wall of the drying chamber.
2.2.2 Particle size and size distribution
Particle size and particle size distributions were measured by Dynamic Laser Scattering
or Photon Correlation Spectroscopy using a Malvern Zetasizer Nano ZS (Malvern
Instruments Ltd, UK). For each sample 3-5mg of spray dried powder were prepared by
suspending the particles in filtered water (0.2μm filter), vortexing and/or sonicating for 2
min if necessary. Each sample was measured in triplicate.
2.2.3 Zeta potential
The zeta potential of the particles was measured using the Zetasizer Nano ZS (Malvern
Instruments Ltd, UK). For that a sample of 3mg of the spray dried nanoparticles was
suspended in 1-2ml of de-ionised water and then vortexed or sonicated before the
measurement. Each measurement was taken in triplicate.
2.2.4 Scanning electron microscope
Surface morphology of spray dried nanoparticles was visualized by scanning electron
microscopy (LEO 1525 Field Emission SEM.). A small amount of nanoparticle powder
was mounted on a brass stub using a double-sided adhesive tape and vacuum-coated with
a thin layer of gold by sputtering.
2.2.5 Drug incorporation
The amount of the hydrophilic drug Isoniazid that was entrapped in the particle powder
after the nanoencapsulation process was measured in triplicate using a
11
spectrophotometric method (UV-Vis, Thermo Spectronic Heliosα). The encapsulation
efficiency of INH in nanoparticles was determined as the mass ratio of the entrapped INH
to the theoretical amount of INH used in the preparation. For that, 50mg of precipitated
particles were re-suspended in 20 ml of deionised water, centrifuged (10
000rpm/10C/5min) to remove the un-encapsulated drug and the supernatant was subject
to UV-Vis Spectrophotometer, read at λ= 262nm for INH assessment. The encapsulated
amount of INH was determined by subtracting INH in the supernatant from total initial
INH amount.
INH stability assessment using HPLC
The stability of INH spray dried powders was assessed by reverse phase-high
performance liquid chromatography-analysis (RP-HPLC) using Shimadzu machine
supplied with Photodiode Array (PDA) detector.
The following characteristics were applied: a Column Phenomenex [(C18 (5μm); (250 x
4.6mm ID)], a mobile phase of 5% (v/v) acetonitrile with 95% (v/v) buffer NaH2PO4 (pH
6.8), at a flow rate of 1 ml/min and at a temperature of 300C. The detection was
performed using PDA at λ= 259nm, on a total injection volume of 20//I.
3. Results and Discussion
All spray drying runs produced nanoparticles with a size ranging from approximately 220
to 800nm. The concentration of the liquid feed did not show any influence on the size of
particles as illustrated with samples where the PVA concentration was changed from 1 to
2%. Only the addition of lactose and Surfynol 104 PG-50 ™ demonstrated a significant
impact on the size and the morphology of nanoparticles. Interestingly, just one drop of
the Gemini surfactant added to the oil phase, drastically reduced the size and the size
distribution of the product, irrespective of either the type of organic solvent or the
concentration of PVA.
During all the sets of experiments beside the temperature, all other parameters of the
spray dryer were kept constant. The mass ratio PLGA: INH (2:1) was also unchanged.
12
The addition of lactose improved significantly the shape of nanoparticles. This effect was
pronounced when dichloromethane was used as organic solvent.
The yields of the powder for all the formulations investigated were in the range of 40-
70%.
The residual water content of selected samples, determined by thermal analysis, showed a
very low level of moisture (-3%).
Results obtained from HPLC indicated the degradation of INH, possibly due to
interaction with lactose. This challenge was overcome by capping the functional groups
of lactose with chitosan, prior to their incorporation in the formulation.
The encapsulation efficiency of INH is approximating 60%.
3.1 Effect of solvent on particles size and morphology
The most commonly used organic solvents in double emulsion technique are
dichloromethane (DCM) and ethyl acetate (EA).
Thus, we decided to monitor the size and the morphology of nanoparticles by varying the
organic solvent. In all cases, when ethyl acetate was used as organic solvent, the
first emulsion obtained presented an aspect of a transient stable emulsion, this
observation being based on the less milky appearance of the emulsion when compared to
the one obtained with DCM.
EξA samples produced very irregular surface morphology compared to samples prepared
with DCM. Particles from EA were highly dimpled and wrinkled before addition of
lactose. Small doughnut-shaped particles were also observed
3.2 Effect of additives
3.2.1 Effect of lactose on particle size and morphology
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The size and the shape as well as surface morphology of nanoparticles were strongly
affected by the composition of the phases. As the initial concentration of lactose was
increased from 5 to 10% w/v, the particles shifted from highly wrinkled to nearly smooth
spheres. The fraction of doughnut-shaped particles decreased sensibly, regardless the type
of solvent used, as depicted by SEM pictures in Fig. 1C and D. However, much more
surface smoothness has been observed with DCM in the scale of observation.
The particle size decreased as we compared with formulations without addition of
lactose, regardless of the type of organic solvent used. The decay was much more
pronounced in case of DCM as illustrated by results presented in Figure 2: the z- average
size of particles dropped from more than 1200 nm to 450nm, when lactose was added to
the formulation.
Zeta potentials were in the positive range because of the presence of chitosan in the
formulation. Its initial concentration was varied between 0.05, 0.1 and 0.3% (w/v) and the
optimisation of the formulation was done with chitosan 0.3%, which resulted in a high
positive zeta potential ~ +45mV.
3.2.2 Effect of Surfynol 104 PG-50 ™ on particle size and yield
Nonionic surfactants, based on acetylenic diol chemistry, represent a unique class of
surfactants providing low surface tension and good de-foaming and surface wetting
characteristics.
Contrary to most surfactants that orient vertically at the water/air interface, the acetylenic
diol surfactants orient horizontally due to their molecular structure. A compact molecule
of this surfactant can migrate very rapidly to the interfacial region providing low values
of the dynamic surface tension (DST). It was reported that for a Surfynol 104 PG-50 ™
bulk concentration of 2.10-6mol. cm-3, the DST dropped around 35 dynes.cm-1. It is,
indeed, this specific property of significantly decreasing the surface tension which
motivated us to select it as a co-surfactant in our formulations.
14
Surfynol 104 PG-50 ™ was added to the internal oil phase before introduction of the drug
aqueous phase. The product obtained was characterised by a very small particle size
about 230nm and the experimental results were reproducible.
The size distribution was equally very narrow (PolyDispersity Index (PDI) ~ 0.1) due
presumably to the capability of Surfynol 104 PG-50 ™ to prevent aggregation.
3.2.3 Effect of PEG and Stearic acid on morphology
It is well established that polyethylene glycol (PEG) is extensively used in drug delivery
strategies in order to generate entities which are less easily recognised by macrophages
and hence exhibit prolonged circulation times in the blood. On the biological level,
coating nanoparticles with PEG sterically hinders interactions of blood components with
their surface and reduces the binding of plasma proteins with PEGylated nanoparticles.
This prevents drug carrier interaction with opsonins and slows down their capture by the
reticulo-endothelial systems (RES).
PEG was introduced together with PVA in the external phase at an initial concentration
of 0.5%w/v, dissolved in de-ionised water.
As we combine the presence of 5ml of PEG (0.5% w/v) in aqueous external phase and
2ml of stearic acid (0.2%w/v) added into the oily phase of the polymer, as a cosurfactant
together with Surfynol 104 PG-50 ™, a significant improvement of the surface
morphology was observed, as depicted in Fig. 3. The reading on Zetasizer provided
smaller particles size of about 270nm with a very narrow distribution (PDI -0.2).
15

We Claim

1. A process for production of nanoparticle carriers for drug delivery, said
nanoparticles being produced by:
- preparing a double emulsion of water-oil-water including one or more polymer
which forms the basis of the nanoparticle carrier;
- blending the drug to be delivered into one of the emulsion phases;
- doping either the oil-phase or the outer-water phase with a carbohydrate;
- doping either the oil-phase or the water phase with a surfactant; and
- spray drying the emulsion to form nanoparticles of a narrow particle size
distribution of 100 nm to 1000 nm.
2. The process as claimed in claim 1, wherein the nanoparticles thus produced are
multifunctional nanoparticles.
3. The process as claimed in claim 1 or claim 2, wherein the carbohydrate is a
saccharide.
4. The process as claimed in claim 3, wherein the saccharide is a disaccharide.
5. The process as claimed in claim 4, wherein the disaccharide is selected from the
group including lactose, maltose, isomaltose, mannobiose, trehalose, and
cellobiose.
6. The process as claimed in any one of claims 3 to 5, wherein the saccharide is
combined with a cationic biodegradable muco-adhesive polysaccharide.
7. The process as claimed in claim 6, wherein the polysaccharide is chitosan and/or
derivatives thereof.
8. The process as claimed in any one of the preceding claims, wherein the surfactant
is a non-ionic surfactant.
9. The process as claimed in claim 8, wherein the surfactant is based on acetylenic
diol chemistry.
16
10. The process as claimed in claim 1, wherein the surfactant is a polymeric non-ionic
surfactant.
11. The process as claimed in claim 10, wherein the polymeric non-ionic surfactant in
the water-phase is PVA.
12. The process as claimed in any one of the preceding claims, wherein there is a
polymer in the oil-phase of the emulsion.
13. The process as claimed in claim 12, wherein the polymer in the oil-phase is
PLGA (poly(lactic-co-glycolic acid)).
14. The process as claimed in any one of the preceding claims, wherein polymers are
present in both the oil-phase and the water-phase.
15. The process as claimed in any one of the preceding claims, wherein the drug is
added to the oil-phase.
16. The process as claimed in claim 15, wherein the drug is a hydrophilic drug which
is added to the internal water-phase.
17. The process as claimed in claim 15 or claim 16, wherein the drug is hydrophobic.
18. The process as claimed in any one of the preceding claims, wherein the drug is
selected from Rifampicin, Isoniazid, Ethambutol, or Pyrazynamide.
19. The process as claimed in any one of the preceding claims, wherein the outer
water-phase of the emulsion includes polyethylene glycol (PEG).
20. The process as claimed in any one of the preceding claims, wherein the oil-phase
includes stearic acid.
21. The process as claimed in any one of the preceding claims, wherein the
nanoparticles thus formed are substantially spherical.
17
22. The process as claimed in claim 21, wherein the particle size distribution of the
nanoparticles is from 180 nm to 250 nm diameter.

Dated this 10th day of August, 2010

Signature

Name: K. RAMA
Agent for the Applicant
Of K&S Partners

To:
The Controller of Patent
The Patent Office, at Chennai