U.S. patent application number 17/559143 was filed with the patent office on 2022-07-21 for nanoparticles and methods for preparation thereof.
The applicant listed for this patent is B.G. Negev Technologies & Applications Ltd. at Ben-Gurion University. Invention is credited to Amnon Sintov.
Application Number | 20220226476 17/559143 |
Document ID | / |
Family ID | 1000006241896 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220226476 |
Kind Code |
A1 |
Sintov; Amnon |
July 21, 2022 |
NANOPARTICLES AND METHODS FOR PREPARATION THEREOF
Abstract
The invention provides a nano-sized particle comprising a
cross-linked polymer, wherein the polymer is selected from the
group consisting of a polyacrylic acid homopolymer; polymethacrylic
acid homopolymer; poly(alkylcyanoacrylate) polymer; a copolymer
comprising at least two monomers selected from acrylic acid,
methacrylic acid, hydroxyethyl acrylate, hydroxyethyl methacrylate,
and alkyl cyanoacrylate/cyanoacrylic acid monomers; carboxymethyl
cellulose; alginic acid polymer, polylactic-polyglycolic acid
(PLGA), and xanthan gum; and wherein said polymer is cross-linked
with a metal ion. A process for preparing such particles is also
provided.
Inventors: |
Sintov; Amnon; (Omer,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
B.G. Negev Technologies & Applications Ltd. at Ben-Gurion
University |
Beer-Sheva |
|
IL |
|
|
Family ID: |
1000006241896 |
Appl. No.: |
17/559143 |
Filed: |
December 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15541555 |
Jul 5, 2017 |
11246934 |
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PCT/IL2016/050004 |
Jan 5, 2016 |
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17559143 |
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62099598 |
Jan 5, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5153 20130101;
A61K 47/32 20130101; A61K 9/5115 20130101; A61K 9/5138
20130101 |
International
Class: |
A61K 47/32 20060101
A61K047/32; A61K 9/51 20060101 A61K009/51 |
Claims
1. A nano-sized particle having an essentially spherical shape and
a size of 50-500 nm, said particle comprising a carbomer
cross-linked with a polyvalent metal ion selected from an alkali
earth metal ion, a divalent transition metal ion, a trivalent metal
ion, or Ag.sup.+ ion.
2. The nano-sized particle according to claim 1, comprising
carbomer cross-linked with a alkali earth metal ion, wherein said
alkali earth metal ion is Ca.sup.2+.
3. The nano-sized particle according to claim 1, wherein a ratio
between said polyvalent metal ion and said carbomer is between 0.01
and 0.5 by weight.
4. The nano-sized particle according to claim 3, wherein said ratio
is between 0.1 and 0.5 by weight.
5. The nano-sized particle according to claim 1, further comprising
at least one active agent.
6. The nano-sized particle according to claim 5, wherein said
active agent is selected from the group consisting of an
antineoplastic agent, an anti-infective agent, and curcumin.
7. The nano-sized particle according to claim 6, wherein said
antineoplastic agent is selected from the group consisting of
doxorubicin, carmustine, fluorouracil, cisplatin, cyclophosphamide,
busulfan, carboplatin, leuprolide, megestrol, lomustine,
levamisole, flutamide, etoposide, cytarabine, mitomycin, nitrogen
mustard, paclitaxel, actinomycin, tamoxifen, vinblastine,
vincristine, thiotepa, and chlorambucil.
8. The nano-sized particle according to claim 6, wherein said
anti-infective agent is selected from the group consisting of an
interferon, acyclovir, valacyclovir, chloramphenicol, gentamycin, a
penicillin, streptomycin, an aminoglycoside, a cephalosporine,
erythromycin, and a tetracycline.
9. The nano-sized particle according to claim 2, further comprising
curcumin.
10. The nano-sized particle according to claim 2, further
comprising chloramphenicol.
11. The nano-sized particle according to claim 2, further
comprising doxorubicin.
12. A pharmaceutical or a cosmetic composition, said composition
comprising the nano-sized particles according to claim 1, and a
pharmaceutically acceptable carrier.
13. The composition according to claim 12, wherein said composition
is pharmaceutical and is formulated to be administered into or
through the skin, optionally subcutaneously, intradermally, or
transdermally.
14. The composition according to claim 12, wherein said composition
is pharmaceutical and is formulated to be administered into or
through a mucosal membrane, optionally orally, buccally, or
intranasally.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/541,555, filed Jul. 5, 2017, which is a
National Stage application of International Patent Application No.
PCT/IL2016/050004, filed on Jan. 5, 2016, which claims priority to
U.S. Patent Application No. 62/099,598, filed on Jan. 5, 2015, each
of which is hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Nanotechnology is becoming increasingly more valuable in the
fields of pharmaceutics and medical engineering, in which it offers
exciting possibilities. This is due to the fact that particles made
of nanoscale can more easily transport with their entrapped drug
molecules through membranal barriers, thus increasing drug
bioavailability. The controlled manner of drug release from the
nanoparticles can prolong the therapeutic efficacy while reducing
local and systemic adverse effects. One of the unmet needs for
nanoparticles (NPs) is chloramphenicol (CHL). Topically-applied CHL
is a widely used antibacterial drug in the form of eye drops for
bacterial conjunctivitis. However, it has a low bioavailability
(1-10%) and short half-life (1.5-5 h), thus needs to be applied
many times a day [1, 2]. CHL is also applied in the vaginal
treatment of local bacterial infections and can be a suitable
candidate for prolonged therapy of vaginitis [3]. In addition, CHL
is recommended by the World Health Organization (WHO) as the first
line treatment of bacterial meningitis, although CHL may cause bone
marrow suppression and aplastic anemia [4]. Several studies [4, 5,
6] have assessed the efficacy of intramuscular long-acting (oily
suspension) CHL for bacterial meningitis and found it as a useful
treatment. Another study using CHL formulation of
polylactide-co-glycolide NPs has demonstrated a high brain uptake
with relatively low accumulation of CHL in bone marrow, indicating
the usefulness of NPs for controlled delivery of the drug [7].
[0003] Numerous methods have been applied to prepare polymeric NPs
for drug delivery. These methods include: (a) emulsion/solvent
evaporation (or emulsification-crosslinking) technology, in which
evaporation of organic solvent (methylene chloride or chloroform)
from an emulsion resulted in calcium-alginate NPs [8], or glycol
chitosan NPs [9]; (b) nanoprecipitation from polymer-drug solution
in water-miscible organic solvent upon slow introduction of
surfactant-containing aqueous phase [10]; (c) salting-out method,
in which a polymer-dissolved organic phase is emulsified in a
saturated salt water under strong stress [11, 12]; (d) spray-drying
procedure, in which dried nanoparticles are created in one-step
process by atomization of polymer solution into a spray droplets
that dried immediately as they contact hot air [13, 14], and (e)
microemulsion as a precursor for NPs production [15-18].
[0004] EP 1943016 discloses composite NPs prepared by collapsing
polymers such as polyacrylic acid, polystyrenesulfonic acid, and
poly(diallyldimethylammonium chloride) from monophase solutions
(aqueous or organic).
[0005] WO 2008051432 describes crosslinking effect by exposing a
discrete portion of ionotropic polymer (e.g., polyacrylic acid) to
a solution of an ionic species which exchanges with an ionic
species in the polymer for production of patterned polymers.
[0006] Nagavarma et al., 2012 [22] summarizes the various
techniques for preparation of NPs at the time of the present
invention; nowadays, salting-out techniques are used with organic
solvents for separation and without organic solvents for
emulsification in order to "minimize stress to protein
encapsulants".
[0007] Therefore, there is still an unmet need for a technique that
would provide the formation of nanoparticles from common,
physiologically acceptable polymers, such as
polyacrylic/methacrylic acid polymers, not known thus far to form
stable, isolated nanoparticles that can carry drugs and other
active agents, wherein the desired technique would not involve the
use of organic solvents and/or other conditions that are not
acceptable in the preparation of encapsulated drugs.
SUMMARY OF THE INVENTION
[0008] In was found in accordance with the present invention that
when an aqueous solution of certain polymers having 50-70% free
carboxylic acid groups or free amino groups, was added, in the
presence of certain surfactants, to an oily phase, a water-in-oil
microemulsion formed, from which self-assembled nanoparticles
emerged by the addition of a cross-linking reagent to the
microemulsion. By using the water-in-oil microemulsion as a reactor
in the presence of a cross linking reagent, and combining the
microemulsion with a salting-out/phase reversal process, a new
method for producing nanoparticles was obtained. This method
enabled formation of nanoparticles from polymers that were not
previously known to assemble into well-defined particles.
[0009] The salting-out/phase reversal process used in accordance
with the present invention eliminates the need to use organic
solvents, such as acetone or alcohol, in any step of the process.
The reactor microemulsion is prepared from pharmaceutically
acceptable components, such as widely-used nonionic surfactants,
and the aqueous polymer solution, for example a carbomer, e.g.
Carbopol.TM. 974P solution, as the inner aqueous phase.
[0010] The involvement of microemulsions in NPs' mode of
preparation is considered a novel composition apart of being a new
process of manufacturing. Incorporation of surfactants and
co-surfactants within a microemulsion (nano-sized emulsion) texture
contributes to diminution and uniformity of NP size, to increasing
of drug loading as well as to controlling of drug release, and
certainly influences their physical properties. The instantly
provided NPs are profoundly different from the particles known in
the art. The particles known as "collapsed polymers" are formed by
a phase separation of polymers in solution thereby converting the
dissolved polymers into globules or other geometric shapes, having
a wide distribution of particle sizes with various degrees of
cross-linking that affect their properties. This undesired
situation is avoided by using polymer-containing nanodroplets
(microemulsions) that shape the desired morphology and geometrics
of the NPs prior to the crosslinking step, as provided for by the
present invention.
[0011] In an additional aspect, the polymeric nanoparticles of the
present invention are slowly and controllably cross-linked with
polyvalent cations, e.g. alkali earth cations. If calcium chloride
solution is used, for example, precipitation of a lump is
immediately generated. The cations may be supplied an organic acid
salts of alkali earth metals with low degree of ionization.
Additionally or alternatively, the cations may be supplied in a
form of microemulsion, as described in greater detail below.
[0012] Therefore, in an aspect of the present invention a
nano-sized particle comprising a cross-linked polymer, wherein the
polymer is selected from the group consisting of a polyacrylic acid
homopolymer; polymethacrylic acid homopolymer;
poly(alkylcyanoacrylate) polymer; a copolymer comprising at least
two monomers selected from acrylic acid, methacrylic acid,
hydroxyethyl acrylate, hydroxyethyl methacrylate, alkyl
cyanoacrylate and/or cyanoacrylic acid monomers; carboxymethyl
cellulose; alginic acid polymer, polylactic-polyglycolic acid
(PLGA), and xanthan gum; and wherein said polymer is cross-linked
with a metal ion. The polymer may further be chemically
cross-linked with a cross-linking moiety selected from the group
consisting of allyl pentaerythritol, allyl sucrose, polyvinyl
alcohol, divinyl glycol, and tetraethylene glycol. Sometimes, the
polymer is a poly(acrylic acid), chemically cross-linked with allyl
pentaerythritol or allyl sucrose. The metal ion may be a polyvalent
metal ion selected from alkali earth metal ions, divalent
transition metal ions, and trivalent metal ions, or Ag+. The alkali
earth metal ion may be Mg.sup.2+, Ca.sup.2+, or Sr.sup.2+. The
particle may further comprise at least one active agent. The active
agent may be selected from curcumin, an antineoplastic agent,
selected from the group consisting of doxorubicin, carmustine,
fluorouracil, cisplatin, cyclophosphamide, busulfan, carboplatin,
leuprolide, megestrol, lomustine, levamisole, flutamide, etoposide,
cytaranine, mitomycin, nitrogen mustard, paclitaxel, actinomycin,
tamoxifen, vinblastine, vincristine, thiotepa, and chlorambucil, an
antiinfective agent selected from the group consisting of
interferons, acyclovir, valacyclovir, chloramphenicol, gentamycin,
penicillin derivatives, streptomycin, aminoglycosides,
cephalosporine, erythromycin and tetracycline. Particularly, the
active agent is chloramphenicol, curcumin or doxorubicin. Sometimes
the particle may have an essentially spherical shape and a size of
50-500 nm. In some particular embodiments, the particle may
comprise poly(acrylic acid) cross-linked with allyl pentaerythritol
or allyl sucrose, and the polyvalent metal ion may be
Ca.sup.2+.
[0013] In an additional aspect, provided is a composition
comprising particles as disclosed herein in a physiologically
acceptable carrier. The composition may be a pharmaceutical
composition or a cosmetic composition.
[0014] In further aspect of the present invention, provided is a
method for producing nano-sized particles, the method comprising
the steps of combining together at least one oil, at least one
surfactant and at least one co-surfactant, to form an oily mixture,
adding a polymer containing carboxylic acid groups to water
optionally in the presence of a base or a buffering agent, to form
a polymer solution, combining together the oily mixture with the
polymer solution to form a water-in-oil microemulsion, adding a
metal ion source into said water-in-oil microemulsion, to form
nano-sized particles, and recovering said nano-sized particles. The
method may further comprise recovering the nano-sized particles by
destabilizing the water-in-oil emulsion to allow phase separation,
followed by collecting the nano-sized particles from the aqueous
phase. Sometimes, the destabilizing is achieved by adding an
aqueous salt solution into the water-in-oil microemulsion
containing the nano-sized particles. The aqueous salt solution may
comprise sodium chloride as the salt, and optionally a
lyophilization additive or an osmolarity adjustment additive.
Sometimes the collecting may be performed by extrusion through a
filter membrane. In some embodiments, the metal ion source may be a
salt selected from salts of alkali earth metals, salts of divalent
transition metals, and salts of trivalent metals, or silver salts.
Particularly, the salt may be Mg.sup.2+, Ca.sup.2+, or Sr.sup.2+
salt. Usually the counter-ion in said metal salt is an organic
acid. In some embodiments, the salt is calcium gluconate. The
polymer may be selected from the group consisting of a polyacrylic
acid homopolymer; polymethacrylic acid homopolymer;
poly(alkylcyanoacrylate) polymer; a copolymer comprising at least
two monomers selected from acrylic acid, methacrylic acid,
hydroxyethyl acrylate, hydroxyethyl methacrylate, alkyl
cyanoacrylate and/or cyanoacrylic acid monomers; carboxymethyl
cellulose; alginic acid polymer; polylactic-polyglycolic acid
(PLGA); and xanthan gum. The polymer may sometimes be chemically
cross-linked with a cross-linking moiety selected from the group
consisting of allyl pentaerythritol, allyl sucrose, polyvinyl
alcohol, divinyl glycol, and tetraethylene glycol. In some
embodiments, the method may further comprise adding an active agent
to the microemulsion and/or to the aqueous polymer solution. The
active agent may be selected from curcumin, a antineoplastic agent
selected from the group consisting of doxorubicin, carmustine,
fluorouracil, cisplatin, cyclophosphamide, busulfan, carboplatin,
leuprolide, megestrol, lomustine, levamisole, flutamide, etoposide,
cytaranine, mitomycin, nitrogen mustard, paclitaxel, actinomycin,
tamoxifen, vinblastine, vincristine, thiotepa, and chlorambucil, an
anti-infective agent selected from the group consisting of
interferons, acyclovir, valacyclovir, chloramphenicol, gentamycin,
penicillin derivatives, streptomycin, aminoglycosides,
cephalosporine, erythromycin, and tetracycline. Sometimes the
surfactant may be at least one of a non-ionic surfactants selected
from the group consisting of capryloylcaproyl macrogol-8-glycerides
(Labrasol), gelatin, albumin, starch, polyvinylpyrrolidone,
polyvinyl alcohol, cetostearyl alcohol, glyceryl monoesters of
fatty acids, polyglyceryl-6-dioleate, polyoxyethyleneglycol
derivatives of fatty acids, polyoxyethyleneglycol ethers,
polyoxyethylene alcohol ethers, polyoxyethylene sorbitan
derivatives, sorbitan esters of fatty acids, and sugar esters.
Sometimes the oil may be selected from the group consisting of
isopropyl palmitate, isopropyl myristate, diethyl sebacate,
diisopropyl adipate, cetyl oleate, oleyl alcohol, hexadecyl
stearate, hexadecyl alcohol, caprylic triglycerides, capric
triglycerides, isostearic triglycerides, adipic triglycerides,
medium chain triglycerides, PEG-6-olive oil (Labrafil), esters of
alkyl or monoglycerides, diglycerides and triglycerides of mono-,
di- or tri-carboxylic acids, propylene glycol myristyl acetate,
lanolin oil, polybutene, wheatgerm oil, vegetable oils, castor oil,
corn oil, cottonseed oil, olive oil, palm oil, coconut oil, canola
oil, sunflower oil, jojoba oil, peanut oil, hydrogenated vegetable
oils, low water-soluble tertiary amides, ethoxylated fats, mineral
oil, petrolatum, animal fats, and polyols. The co-surfactant may be
selected from the group consisting of propylene carbonate,
propylene glycol, tetraglycol (glycofurol), a polyethylene glycol,
benzyl alcohol, propanol, and butanol. Particularly, the active
agent may be chloramphenicol, curcumin or doxorubicin. More
particularly, the polymer may be poly(acrylic acid) cross-linked
with allyl pentaerythritol or allyl sucrose, and the polyvalent
metal ion may be Ca.sup.2+, and the organic acid salt may be
gluconate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B show pseudo-ternary phase diagrams
representing a microemulsion system made of isopropyl palmitate,
glyceryl oleate, and Labrasol, propylene carbonate, and water.
[0016] FIGS. 2A, 2B, and 2C show TEM and SEM scans of
nanoparticles.
[0017] FIGS. 3A and 3B show the effect of Ca.sup.2+ gluconate level
on average particle size.
[0018] FIG. 4 shows the effect of aqueous phase content of the
microemulsion on the average particle size of CHL-loaded and
unloaded nanoparticles.
[0019] FIG. 5 shows the effect of aqueous phase content of the
microemulsion on CHL loaded in the nanoparticle dispersion produced
by 0.086% and 0.129% Ca.sup.2+ gluconate concentration.
[0020] FIGS. 6A and 6B show (A) a plot of In[C_1 0 V_1-C_2
(t)(V_2+V_1)] vs dialysis time of four different concentrations of
chloramphenicol in NaCl 0.9% w/v; and (B) chloramphenicol recovery
and the apparent permeability constant (k') values obtained from
dialysis of various chloramphenicol concentrations in NaCl 0.9% w/v
(V_1:donor solution,V_2:receiver solution).
[0021] FIG. 7 shows drug release from drug-loaded
nanoparticles.
[0022] FIGS. 8A, 8B, and 8C show the dependence between the
nanoparticle radius (R, given in nm) and the aqueous content,
designated "AqC" (8A), amount of polymer, designated "Pol." (8B),
and the amount of cross-linker, expressed in molar percent,
designated "Ca.sup.+2" (8C).
[0023] FIGS. 9A and 9B show the shape of nanoparticles loaded with
curcumin in TEM scans.
[0024] FIG. 10 shows the reference curve obtained by potentiometric
titration of three different polymer amounts is exemplified, from
pH 4.5 to 9.0.
[0025] FIG. 11 shows the release behavior of curcumin from hydrogel
nanoparticles with drug loading of 0.48 w/w at 37.degree. C. at pH
7 with iBSA (2% w/v) and NaCl (0.9% w/v) added to the dissolution
medium.
[0026] FIG. 12A and 12B show graphs of cumulative release for
doxorubicin from nanoparticles.
[0027] FIGS. 13A, 13B, and 13C show results indicating that NPs
loaded with anticancer drugs are more efficacious than the drugs in
plain solutions. FIG. 13A shows that DOX-loaded NPs are more
cytotoxic than DOX in plain solution (24 h incubation). FIG. 13B
demonstrates the same phenomenon with curcumin. FIG. 13C presents
dose response behavior of DOX-loaded and unloaded NPs vs. solutions
at various concentrations after 24-h and 48-h incubation.
[0028] FIGS. 14 A and 14B show (A) the tumor growth inhibition by
the chemotherapeutic drug in terms of volume change during the
course of treatment (18 days) and (B) the effect on mortality
[0029] FIG. 15 shows a schematic representation of the method for
producing nano-sized particles according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] In one aspect, the present invention provides nano-sized
particles comprising a polymer having 50-70% mole free carboxylic
acid groups. Preferably, the polymer has not been known hitherto to
form nanoparticles. The nanoparticles may further comprise at least
one drug. Preferably, the polymer is a pharmaceutical-grade
polymer.
[0031] The polymers particularly suitable for formation of the
nanoparticles of the present invention include homopolymers of
acrylic acid and methacrylic acid, copolymers comprising monomers
of acrylic acid, methacrylic acid and/or their hydroxyalkyl, e.g.
hydroxyethyl, monomers, copolymers comprising monomers of alkyl
cyanoacrylate and/or acrylic/methacrylic acid/or cyanoacrylic acid,
cellulose derivatives, e.g. carboxymethyl cellulose or xanthan gum,
and alginic acid.
[0032] Sometimes, the polymer forming the nanoparticles is a cross
linked polymer in itself. The cross-links may by a moiety selected
from, but not limited to, a polyalkenyl polyether, allyl sucrose,
polyvinyl alcohol, divinyl glycol, or tetraethylene glycol. In
particular embodiments, the polymer, for example, a polyacrylic
acid polymer or polymethacrylic acid polymer, is cross-linked with
a polyalkenyl polyether moiety such as allyl ethers of
pentaerythritols, or polyhydric alcohols containing more than one
alkenyl group per molecule and at least 2 hydroxyl groups.
[0033] In certain embodiments, the nano-sized particles of the
invention are formed from a cross-linked poly acrylic acid polymer
(carbomer), preferably a polyacrylic acid polymer cross-linked with
allyl pentaerythritol or allyl sucrose.
[0034] The term "carbomer" as used herein relates to a homopolymer
of acrylic acid, which is cross-linked with any of several
polyalcohol allyl ethers and/or polyalkenyl ethers. As used herein,
"carbomer" also encompasses "carbopol", which is a polymer of
acrylic acid cross-linked with polyalkenyl ethers or divinyl
glycol. Carbomers are commonly used in stabilizing emulsions and
providing viscosity to solutions. Carbopol.RTM. homopolymers are
known as "carboxyvinyl polymers" and "carboxy polymethylene
polymers". Examples of commercially available carbopols include the
carboxy polymethylene Carbopol.RTM. 934P and the polyacrylic acid
Carbopol.RTM. 980NF. Carbopol copolymers, such as Carbopol.RTM.
1342 NF and 1382, which are acrylates/Alkyl Acrylates cross
polymers are also encompasses by the term "carbomer".
[0035] Carbopol polymers are manufactured, starting from primary
polymer particles of about 0.2 to 6.0 micron average diameter, by a
cross-linking process. Depending upon the degree of cross-linking
and manufacturing conditions, various grades of Carbopol are
available. Carbopol.RTM. 934P is cross-linked with allyl sucrose
and is polymerized in solvent benzene. Carbopol.RTM. 71G, 971P,
974P are cross-linked with allyl penta erythritol and polymerized
in ethyl acetate. Polycarbophil is cross-linked with divinyl glycol
and polymerized in solvent benzene. Carbopol.RTM. 971P has slightly
lower level of cross-linking agent than Carbopol.RTM. 974P although
they are both manufactured by the same process under similar
conditions. Carbopol 71G is the granular form Carbopol grade.
[0036] The polyalcohol portion gives a carbomer its high water
solubility. Carbomers readily absorb water, get hydrated and swell.
They are capable of absorbing large amounts of water, increasing in
volume up to 1,000 times their original volume and 10 times their
original diameter, and most often form a gel when exposed to a pH
environment above 4.0 to 6.0. The carboxyl groups provided by the
acrylic acid backbone of the polymer are responsible for many of
the product benefits. Because the pKa of these polymers is 6.0 to
0.5, these carboxyl ionize, resulting in repulsion between the
native charges, which adds to the swelling of the polymer. In some
cases the swollen polymer chains form gels and thick solutions. The
flocculated agglomerates and colloidal, mucilage-like dispersions
formed when a carbomer absorbs water cannot be broken into the
ultimate particles once it is produced, and can be viewed as a
network structure of polymer chains interconnected via
cross-linking. The readily water-swellable carbomer polymers are
thus used in a diverse range of pharmaceutical applications mostly
as excipients.
[0037] Preferably, the microemulsion has droplets size of between
5-50 nm.
[0038] The microemulsion reactor containing the (cross linked)
polymer, for example carbomer, may be a coacervate formed by the
coacervation process. The term "coacervation", as used herein,
relates to a colloidal phenomenon, wherein a colloidal solution
separates into two non-miscible parts, one richer in dispersed
colloidal material than the other. The colloid-rich phase is called
"the coacervate". The colloid-poor phase is called the equilibrium
liquid and has a low or negligible content of colloidal material.
In a coacervate, the distribution of colloid particles is
statistically uniform, as in the original solution, although their
concentration may be increased. In the water-in-oil microemulsion
resulting from combining an oily mixture with an aqueous solution
of a polymer, the spontaneously formed microdroplets of polymer
solution may form coacervates.
[0039] The nano-sized particles provided by the present invention
are soft, flexible particles having spherical shape and a size of
50-500 nm. Preferably, the particles have size of 100-400 nm, most
preferably 200-300 nm, but sometimes the particles may be 50-200
nm, and preferably 50-100 nm.
[0040] The polymers suitable for the present invention may be
cross-linked with a polyvalent cation. The cations suitable for the
present invention are polyvalent cations. These include alkali
earth cations, such as Ca.sup.2+, Mg.sup.2+ and Sr.sup.2+; divalent
transition metals cations, such as Fe.sup.2+, Mn.sup.2+, Cu.sup.2+,
Mo.sup.2+ and Zn.sup.2+; and trivalent cations, such as Cr.sup.3+,
Au.sup.3+, Fe.sup.3+, and Al.sup.3+. Additionally or alternatively,
the cation may form coordination bonds with structures in the
polymer, e.g. with carbonyls, in addition to ionic interactions,
e.g. transition metals' cations, and Ag.sup.+ and Au.sup.+.
[0041] The crosslinking ratio between the polyvalent cation and the
polymer in the nanoparticle may be adjusted to achieve the desired
amount of cross-links. Usually, the cross-linking ratio is between
0.01 to 0.5 of cation ion weight per polymer weight, and more
preferably between 1:10 to 1:1. Sometimes, the crosslinking ratio
may be provided as percentage of equivalents, taking into account
the polyvalent nature of the cations. In such cases, the
cross-linking ratio may be from 0.01 to 0.1, and preferably between
1:10 to 1:2.
[0042] The nano-sized particles of the invention are suitable as a
platform for carrying at least one active agent. The active agent
may be encapsulated within the nano-sized particle, non-covalently
associated thereto, ionically or electrically associated thereto,
and/or embedded or incorporated in the nano-sized particle
matrix.
[0043] In certain embodiments, the active agent is encapsulated
within the nano-sized particle as a well-defined core inside the
nanoparticle, or as a gradient from inside to the boundary of said
nanoparticle.
[0044] In certain other embodiments, the active agent is embedded
within the nano-sized particle matrix either homogeneously by being
uniformly dissolved in the polymer matrix, or heterogeneously by
being dispersed as clusters or aggregates in the polymer
matrix.
[0045] Any active agent may be delivered or carried by the
nanoparticles of the invention. Particular classes of active agents
include, but are not limited to, biologically active agents,
radioactive labels, diagnostic markers, cosmetic or cosmeceutic
agents, nutrition or naturaceutic agents, as well contrast agents
for imaging procedures.
[0046] Non-limiting examples of biologically active agents in the
nano-sized particles include curcumin, antineoplastic agents, such
as doxorubicin, carmustine, fluorouracil, cisplatin,
cyclophosphamide, busulfan, carboplatin, leuprolide, megestrol,
lomustine, levamisole, flutamide, etoposide, cytaranine, mitomycin,
nitrogen mustard, paclitaxel, actinomycin, tamoxifen, vinblastine,
vincristine, thiotepa, chlorambucil, antiinfective (e.g. antiviral
or antibacterial) agents such as interferons (e.g.,
alpha2a,b-interferon, beta-interferon), acyclovir, valacyclovir,
chloramphenicol, gentamycin, penicillin derivatives, streptomycin,
aminoglycosides, cephalosporine, erythromycin and tetracycline.
[0047] In certain embodiments, the present invention provides
nano-sized particles loaded with chloramphenicol, curcumin, or
doxorubicin (also known as Adriamycin). From the pharmacology
standpoint, the nanoparticles of the present invention fulfill the
requirements of ideal drug delivery systems: ease and
reproducibility of preparation, ease of storage and administration
in a sterile form, satisfying drug-loading capacity, low toxicity,
and feasibility for scale-up production. By varying one or more
experimental parameters (polyvalent ions and their quantities,
polymer concentration, water content, surfactants ratio, etc.),
various types of nanoparticles can be obtained, each exhibiting
specific features regarding the nature of the drug and the way it
is encapsulated.
[0048] In particular embodiments, the chloramphenicol-containing
nano-sized particles, or the curcumin-containing nano-sized
particles of the invention are comprised of a cross-linked
polyacrylic acid polymer (carbomer). In more particular
embodiments, the carbomer is polyacrylic acid cross-linked with
allyl pentaerythriol or allyl sucrose.
[0049] The nano-sized particles of the invention may further
comprise at least one additive for targeting purposes, for
enhancing permeability and/or for increasing the stability of the
nano-sized particle. Non-limiting examples of additives for
nanoparticle targeting include paramagnetic moieties such as iron
oxide. Magnetic targeting combined with ultrasound directed to the
diseased area (tumor, infection, or inflammation) can mediate a
more effective and safer drug delivery.
[0050] In another aspect, the present invention provides a
composition comprising the nano-sized particles of the invention
and a physiologically acceptable carrier. In certain embodiments,
the composition is a pharmaceutical composition. In certain other
embodiments, the composition is a cosmetic composition.
[0051] In a further aspect, the present invention provides a method
for producing nano-sized particles, the method comprising the steps
of combining together at least one oil, at least one surfactant and
at least one co-surfactant, to form an oily mixture, adding a
polymer containing carboxylic acid groups to water optionally in
the presence of a base or a buffering agent, to form a polymer
solution, combining together the oily mixture with the polymer
solution to form a water-in-oil microemulsion, adding a metal ion
source into said water-in-oil microemulsion, to form nano-sized
particles, and recovering said nano-sized particles. The method may
further comprise recovering the nano-sized particles by
destabilizing the water-in-oil emulsion to allow phase separation,
followed by collecting the nano-sized particles from the aqueous
phase. Sometimes, the destabilizing is achieved by adding an
aqueous salt solution into the water-in-oil microemulsion
containing the nano-sized particles.
[0052] Preferably, the collecting of the nanoparticles is performed
by extrusion through a filter membrane of suitable pore size, e.g.
100-200 nm pore size.
[0053] The method of the present invention may further comprise at
least one step of: [0054] a. adding the active agent, optionally
dissolved in an aqueous solution, to the microemulsion and/or to
the aqueous polymer solution; [0055] b. providing the cross-linking
reagent in a microemulsion of the part of the oily mixture and
water.
[0056] Non-limiting examples of the active agents for use in the
method include curcumin, antineoplastic agents, such as
doxorubicin, carmustine, fluorouracil, cisplatin, cyclophosphamide,
busulfan, carboplatin, leuprolide, megestrol, lomustine,
levamisole, flutamide, etoposide, cytaranine, mitomycin, nitrogen
mustard, paclitaxel, actinomycin, tamoxifen, vinblastine,
vincristine, thiotepa, chlorambucil, antiinfective (e.g. antiviral
or antibacterial) agents such as interferons (e.g.,
alpha2a,b-interferon, beta-interferon), acyclovir, valacyclovir,
chloramphenicol, gentamycin, penicillin derivatives, streptomycin,
aminoglycosides, cephalosporine, erythromycin and tetracycline.
[0057] In certain embodiments, the polymers used in the method of
the invention are polymers containing 50 to 70% mol free carboxylic
acid groups. In preferred embodiments, the polymers are selected
from cross-linked polymers of acrylic acid and/or methacrylic acid,
alginic acid polymers, xanthan gum, polymers of lactic acid and/or
glycolic acid. In particular embodiments, the polymer is a
cross-linked polymer of acrylic acid and/or methacrylic acid as
defined above.
[0058] In certain other embodiments, the polymers used in the
method of the invention are polymers containing free amino groups.
In preferred embodiments, the polymers are selected from cationized
guar gums or polyquaternium polymers.
[0059] In those embodiments wherein the method of the invention is
utilized for the production of nanoparticles of polymers with high
content of free amino groups, the cross linking reagent most
preferably employed is a carboxylic acid compound such as citric
acid, boric acid, or phosphoric acid, preferably citric acid.
[0060] The oil employed in the method provided herein is selected
from at least one of isopropyl palmitate, isopropyl myristate,
diethyl sebacate, diisopropyl adipate, cetyl oleate, oleyl alcohol,
hexadecyl stearate, hexadecyl alcohol, caprylic triglycerides,
capric triglycerides, isostearic triglycerides, adipic
triglycerides, medium chain triglycerides (C.sub.8-C.sub.10 fatty
acids), PEG-6-olive oil (Labrafil), esters of alkyl or
monoglycerides, diglycerides and triglycerides of mono-, di- or
tri-carboxylic acids, propylene glycol myristyl acetate, lanolin
oil, polybutene, wheatgerm oil, vegetable oils such as castor oil,
corn oil, cottonseed oil, olive oil, palm oil, coconut oil, canola
oil, sunflower oil, jojoba oil, peanut oil, and hydrogenated
vegetable oils, low water-soluble tertiary amides, ethoxylated
fats, mineral oil, petrolatum, animal fats, and polyols. The
preferred oil is isopropyl palmitate or isopropyl myristate.
[0061] The surfactants employed in the method of the invention are
preferably non-ionic surfactants such as, but not limited to, at
least one of capryloylcaproyl macrogol-8-glycerides (Labrasol),
gelatin, albumin, starch, polyvinylpyrrolidone, polyvinyl alcohol,
cetostearyl alcohol, glyceryl monoesters of fatty acids (e.g.,
glyceryl monostearate, glyceryl monooleate, or glyceryl dioleate),
polyglyceryl-6-dioleate (Plurol oleique), polyoxyethyleneglycol
derivatives of fatty acids (e.g., Myrj 45, 49, 51, 52, 52S, 53,
59), polyoxyethyleneglycol ethers (e.g., polyoxyethylene (23)
dodecyl ether or Brij 35), polyoxyethylene alcohol ethers,
polyoxyethylene sorbitan derivatives (polysorbates, e.g., Tweens
such as Tween 20, 40, 60, 80, 85), sorbitan esters of fatty acids
(e.g., sorbitan sesquioleate, sorbitan isostearate, sorbitan
monolaurate, sorbitan monostearate, sorbitan monooleate), and sugar
esters (e.g., Sisterna sucrose esters, which are based on sucrose
and vegetable fatty acids). In some embodiments, at least two
surfactants are used in the preparation of the oily mixture. The
preferred surfactants are capryloylcaproyl macrogol-8-glycerides
(Labrasol) and glyceryl monoesters of fatty acids.
[0062] The suitable co-surfactants for the method of the present
invention are selected from at least one of propylene carbonate,
propylene glycol, alkanols, e.g. propanol or butanol, benzyl
alcohol, tetraglycol (glycofurol), and polyethylene glycol.
[0063] The ratio between the co-surfactants and the surfactants may
be between 0.1 and 0.9, preferably between 0.2 and 0.6.
[0064] The polymer is dissolved in water to furnish polymeric
solution. The polymeric solution may contain from 0.001% wt of
polymer to about 0.5% wt, preferably from 0.01% wt to 0.05% wt.
[0065] Bases or buffering agents may be used in the preparation of
the polymer solution for the method of the present invention. These
include, but not limited to, alkali hydroxides, e.g. sodium or
potassium hydroxide, ammonium hydroxide, suitable amines, e.g.
triethyl amine, and triethanolamine.
[0066] Combining of the oily mixture with the polymer solution
results in a spontaneous formation of the microemulsion. The
combining may be performed at any suitable temperature, e.g. at
room temperature, from about 15.degree. C. to about 30.degree. C.,
but may also be performed from 0.degree. C. to 100.degree. C.,
preferably about 30.degree. C.
[0067] Cross-linking agent is added to the formed microemulsion to
effect cross-linking. The cross-linking agent may usually be a
polyvalent cation as described herein. The polyvalent cation is
usually supplied in a form of a salt, preferably a salt of an
organic acid that dissociates slowly in water. The cation
counter-ions may be a saccharidic acid ion, e.g. gluconate,
picolinic acid ion, or an aliphatic acid ion. Generally, the
counter-ion may have a pK.sub.a of between 2 and 7. Preferably, the
counter-ion is gluconate. Most preferably, the cross-linking agent
is calcium gluconate.
[0068] Sometimes, the cross-linking agent may be provided as a
microemulsion. The microemulsion may preferably contain similar or
same composition of the oily mixture that the polymer water-in-oil
microemulsion. The aqueous phase naturally comprises the
cross-linking agent in aqueous solution, as disclosed herein.
[0069] Combining the cross-linking agent with the microemulsion
requires a meticulous control over the addition rate of the
cross-linking agent. Usually the addition rate is selected to
prevent polymer precipitation as a lump, and may be selected from
0.001 mL/min/mL of microemulsion, to 0.5 mL/min/mL of
microemulsion, more preferably 0.03 mL/min/mL of microemulsion.
[0070] The cross-linked nano-particles may be allowed to maturate
for 5-30 minutes.
[0071] The nanoparticles are extracted into aqueous medium from the
microemulsion by reversing the phase of the emulsion. Adding
sufficient amount of aqueous phase causes phase separation and
extracts the nanoparticles into the aqueous phase. Usually, the
additional aqueous phase is added in amount of about 1:1 with the
microemulsion, and may be between 1:2 and 2:1, preferably 1:1.
[0072] The aqueous solution for phase reversal usually contains
salts, e.g. sodium chloride, or any water soluble salt, e.g. KCl,
NaHCO.sub.3, Na.sub.3PO.sub.4, NaH.sub.2PO.sub.4, and Na2HPO4. The
salts may be in the amount varying from 0.5% wt to 10% wt;
preferably the salt is sodium chloride and the concentration is
between 3-6%. The aqueous solution may further comprise at least
one additive. The additive may be used in further processing of the
nanoparticles, such as lyophilization, or may be a part of the
pharmaceutical composition. Such additives include, but not limited
to, mannitol, and lactose. The additives may be employed in
concentrations between 0.01 and 1%, preferably 0.1%.
[0073] Upon addition of the aqueous solution the phase separation
will occur. The phase separation may take from 15 minutes to 4-12
hours, and may be performed at 25.degree. C. to 50.degree. C.,
preferably between 15.degree. C. and 35.degree. C. The phase
separation may also be assisted by instrumental means, such as
centrifugation, elevated or reduced pressure. In this case the
phase separation may take less time.
[0074] Collecting of the obtained nanoparticles may be performed by
a variety of methods, e.g. centrifugation, filtration through a
filter of a suitable size, dialysis and others as known in the
art.
[0075] The active agent used in the method of the invention may be
any of the aforementioned active agents, namely, a biologically
active agent, a radioactive label, a contrast agent, diagnostic
marker, a cosmetic or cosmeceutic agent, a nutrition or
naturaceutic agent. The active agent may be encapsulated within the
nano-sized particle, non-covalently associated thereto, ionically
or electrically associated thereto, and/or embedded or incorporated
in the nano-sized particle matrix, preferably encapsulated within
the nano-sized particle as a well-defined core inside the
nanoparticle or as a gradient from inside to the boundary of said
nanoparticle. When embedded within the nano-sized particle matrix,
the active agent may either be uniformly dissolved in the polymer
matrix (homogeneously embedded) or dispersed as clusters or
aggregates in the polymer matrix (heterogeneously embedded).
[0076] In certain embodiments, nano-sized particles containing
chloramphenicol, doxorubicin or curcumin are obtained by the method
of the invention. In preferred embodiments, these nano-sized
particles comprise a cross-linked polyacrylic acid polymer,
preferably an allyl pentaerythriol-cross-linked polyacrylic acid
polymer (a carbomer), and are formed by the use of the
cross-linking reagent calcium gluconate.
EXAMPLES
Materials and Methods
1. Materials
[0077] Isopropyl palmitate, propylene carbonate and calcium
gluconate were purchased from Aldrich (Sigma-Aldrich Inc., St.
Louis, Mo.). Glyceryl oleate was obtained from Uniqema, Bromborough
Pool, The Wirral, UK. Labrasol was obtained from Gattefosse,
France. Carbopol.RTM. 974P NF Polymer was obtained from Lubrizol,
Ohio, USA. High-performance liquid chromatography (HPLC) grade
solvents were obtained from J. T. Baker (Mallinckrodt Baker, Inc.,
Phillipsburg, N.J.). Chloramphenicol was obtained from Sigma,
Rehovot, Israel. Curcumin was obtained from Sigma, Rehovot, Israel.
Doxorubicin was obtained from Euroasian Chemicals Pvt. Ltd.
(Mumbai, India).
2. NPs Preparation
[0078] Generally, microemulsions were prepared by mixing Labrasol,
glyceryl oleate (surfactants) and isopropyl palmitate (oil) with
propylene carbonate (co-surfactant). The co-surfactant to
surfactants (CoS/S) weight ratio was 1:5, and the surfactants'
ratio was 1:3. Appropriate volumes of Carbopol.RTM. 974P NF
solution (pH=7) were solubilized along a dilution line--DL20, (DL20
means a line representing a surfactants-to-oil ratio of 80:20)
(FIG. 1A). Formulations prepared along DL20 contained 0.01 wt %
polymer in the microemulsion, and this concentration was kept
constant regardless of changing in water content. The monophasic
formulations were formed spontaneously at room temperature. After
the microemulsion had been loaded with CHL and a clear liquid was
obtained, an aqueous solution of calcium gluconate was added. To
examine the calcium effect on NP size, several calcium gluconate
levels were introduced into microemulsions to make up a total water
phase of 25% (and 0.01% polymer concentration) at the following
concentrations: 0.043%, 0.086%, 0.129%, 0.258%, and 0.430%.
[0079] The addition of calcium gluconate was performed by using a
peristaltic pump operated at a controlled rate of 0.3 ml/min while
microemulsions were kept at 35.degree. C. under continuous stirring
(300 rpm).
[0080] Turning now to FIGS. 1A and 1B, pseudo-ternary phase diagram
represents a microemulsion system (shaded area) made of isopropyl
palmitate (oil), glyceryl oleate and Labrasol (as surfactants at a
1:3 w/w ratio), propylene carbonate (co-surfactant) and water. The
co-surfactant/surfactants ratio was 1:5. Lines DL20 and DL12
represent water dilution line at a constant surfactant-to-oil ratio
of 4 (w/w) and 88:12, respectively.
[0081] Similarly, the microemulsion was prepared by mixing
Labrasol, glyceryl oleate (surfactants) and isopropyl palmitate
(oil) with propylene carbonate (co-surfactant) [20]. The
co-surfactant to surfactants (CoS/S) weight ratio was 1:5, and the
surfactants' ratio was 1:3. Appropriate volumes of Carbopol.RTM.
974P NF solution (pH=7) were solubilized along a dilution
line--DL12, (DL12 means a line representing a surfactants-to-oil
ratio of 88:12) (FIG. 1B). Formulations prepared along DL12 usually
contained 0.01wt % polymer in the microemulsion, and this
concentration was kept constant regardless of water content. The pH
7.5 was adjusted by a 0.5% triethanolamine solution. The monophasic
formulations were formed spontaneously at room temperature. After
the microemulsion had been loaded with 400 .mu./ml doxorubicin and
a clear colored liquid was obtained, an aqueous solution of calcium
gluconate was added under constant stirring (1200 rpm) using a
10-ml syringe pump operated at a controlled rate of 0.3 ml/min at a
temperature range of 25.degree. C.-35.degree. C. To examine the
cross-linking effect of calcium ions on NP size and release,
several calcium gluconate concentrations were introduced into the
microemulsion (16% w/w of total aqueous phase, and 0.01% polymer
concentration were kept constant) at the following Ca++
ions/polymer ratios: 1:10, 3:10, 1:2, and 1:1, as described below.
To examine the effect of polymer concentrations on the size and
release of the resulted NPs, three concentrations were
prepared--0.01, 0.02, and 0.04%--while the Ca++ ions/polymer ratio
(3:10) and the microemulsion's aqueous phase (16%) were kept
constant. To examine the effect of the aqueous phase in the
microemulsion on the NPs, formulations containing 16%, 20% and 25%
aqueous phase were prepared, while the polymer concentration and
the Ca++ ions/polymer ratio were kept constant.
3. Salting-Out and Separation of NPs
[0082] A solution containing 0.9% (w/v) sodium chloride was mixed
gently with the calcified microemulsion at a 1:1 ratio, and the
mixture was poured into a burette and allowed to separate for 12
hours. Two phases with different densities were obtained. The lower
phase, a clear aqueous fluid containing the drug-loaded NPs in
dispersion, was collected and extruded through a membrane of 200 nm
(in diameter) pore size using Avanti Mini-Extruder (Avanti Polar
Lipids Inc, Alabama, USA).
4. Size and Microscopic Analysis
4.1 Dynamic Light Scattering (DLS)
[0083] The hydrodynamic diameter spectrum of the NPs was collected
using CGS-3 Compact Goniometer System (ALV GmbH, Langen, Germany).
The laser power was 20 mW at the He--Ne laser line (632.8 nm).
Correlograms were calculated by ALV/LSE 5003 correlator, which were
collected at 60.degree., during 10 s for 20 times, at 25.degree. C.
The NP size was calculated using the Stokes-Einstein relationship,
and the analysis was based on regularization method as described by
Provencher [19].
4.2 Scanning Electron Microscopy (SEM)
[0084] The surface morphology of the NPs was inspected by scanning
electron microscopy (SEM, JEOL JSM-35CF). The NP dispersion was
first lyophilized and then coated with an ultrathin (100 .ANG.)
layer of gold in a Polaron E5100 coating apparatus. The samples
were viewed under SEM at an accelerating voltage of 25 kV.
4.3 Transmission Electron Microscopy (TEM)
[0085] TEM images were recorded on a JEOL JEM-1230 transmission
electron microscope (JEOL LTD, Tokyo, Japan) operating at 120 kV.
Samples of NP dispersions were deposited on a copper 300 mesh grid,
coated with Formvar and carbon (Electron Microscopy Sciences, Fort
Washington, Pa., USA) and allowed to stand for 1 minute after which
any excess fluid was adsorbed in a filter paper. Subsequently, one
drop of 1% phosphotungstic acid (PTA) solution was applied on the
grid and allowed to dry for 1 minute. Electron micrographs were
taken using TemCam-F214 (Tietz Video & Image Processing Systems
(TVIPS), Gauting, Germany).
5. Determination of Drugs in NP Dispersion
5.1. Chloramphenicol
[0086] Aliquots of 20 l from each sample were injected into a HPLC
system, equipped with a prepacked column (Luna C18 column, 5 .mu.m,
150 mm.times.4.6 mm, Phenomenex, Torrance, Calif.). The HPLC system
(Shimadzu VP series) consisted of an auto-sampler and a diode array
detector. The quantification of chloramphenicol was carried out at
275 nm. The samples were chromatographed using an isocratic mobile
phase consisting of acetonitrile-phosphate buffer, pH 5 (30:70) at
a flow rate of 1 ml/min.
[0087] The drug concentration in the NP dispersion (lower phase)
was estimated according to the following equation:
C dis = Q T - W u .times. p * C u .times. p W l .times. o .times. w
##EQU00001##
where Q.sub.T is the total amount of the drug dissolved in the
microemulsion, W.sub.up and W.sub.low are the weights of the upper
and lower phases, respectively and C.sub.up is the concentration of
the drug in the upper phase (wt/wt).
5.1. Curcumin
[0088] Aliquots of 20 .mu.L from each sample were injected into a
HPLC system, equipped with a prepacked column (Reprosil-Pur 300
ODS-3, 5 um, 250 mm.times.4.6 mm, Dr. Maisch, Germany) which was
constantly maintained at 30.degree. C. The HPLC system (Shimadzu VP
series) consisted of an auto-sampler and a diode array detector.
The quantification of curcumin was carried out at 425 nm. The
samples were chromatographed using a mobile phase consisting of
acetonitrile-35 mM acetic acid (80:20) at a flow rate of 1 ml/min.
A calibration curve (peak area vs. curcumin concentration) was
constructed by running standard curcumin solutions in methanol. The
calibration curves were linear over the range 0.025-10 .mu.g/ml
(r>0.99).
5.3. Doxorubicin
[0089] Aliquots of 20 .mu.l were injected into a HPLC system,
equipped with a prepacked column (ReproSil-Pur 300 ODS-3, 5 .mu.m,
150 mm.times.4.6 mm, Dr. Maisch, Germany), which was constantly
maintained at 30.degree. C. The HPLC system (Shimadzu VP series)
consisted of an auto-injector and a photodiode array detector. The
quantification of DOX was carried out at 234 nm. The samples were
chromatographed using an isocratic mobile phase consisting of
acetonitrile-0.2% heptanesulfonic solution (40:60) at a flow rate
of 1 ml/min.
6. Drug Release Studies
[0090] The amount of drug released from the NPs was determined by a
dynamic dialysis technique monitoring the drug concentration in the
receiver solution (i.e., outer solution surrounding a dialysis
bag). The method is based on the assumption that drug is first
released from the NPs into the donor solution (i.e., the
dissolution medium inside the dialysis bag). Subsequently, the drug
can diffuse through the dialysis bag from the donor to the
receiver.
[0091] A sample of 3 ml of the NP dispersion (1:1 diluted with
saline) was introduced into a dialysis bag (SnakeSkin Dialysis
Tubing, 10K MWCO, 22 mm, Thermo Fisher Scientific, Rockford, USA).
The sample was dialyzed against 60 ml of saline solution (NaCl
solution, 0.9%, w/v) for 7 hours. The receiver solution was
agitated by magnetic stirring throughout the experimental
procedure. Samples of 200 .mu.l were withdrawn from the receiver
solution at predetermined time intervals and their drug
concentrations were measured by HPLC.
Example 1: Production and Isolation of Calcium-Carbopol
Nanoparticles
[0092] Self-assembled polymeric NPs were engineered by using a
water-in-oil microemulsion template containing water-soluble
polymer in the inner aqueous phase. The polymer (Carbopol 974P) in
the nanodroplets was crosslinked by Ca.sup.+2 ions to form NPs
which were separated by simple and direct salting-out. Production
of NPs by salting-out was previously described [E. Allemann, J. C.
Leroux, R. Gurny, E. Doelker, In vitro extended-release properties
of drug-loaded poly(DL-lactic acid) nanoparticles produced by a
salting-out procedure, Pharm. Res. 10 (1993) 1732-1737] for
poly(DL-lactic acid) NPs. In this publication, the polymer was
dissolved in an organic phase (acetone), which should be miscible
in all proportions with pure water but being separated and
emulsified in salt-containing water. According to this method, NPs
were generated upon gradual dilution of the emulsion to create a
monophasic system. We utilized this method for calcium-Carbomer NP
production, however, we have employed the salting-out technique for
separation of NPs from the microemulsion oily components rather
than for generation of NPs. This salting-out technique has been
utilized in this research to avoid separation and washing of NPs by
organic solvents.
Specific Example 1.1--Neat Calcium-Carbomer Nanoparticles
[0093] More specifically, the process was performed as follows.
[0094] An oil phase was produced by mixing together isopropyl
palmitate, 6.0 g; glyceryl oleate, 9.25 g; Labrasol, 27.75 g; and
propylene carbonate, 7.4 g; to furnish 50.4 g of the oil phase. The
materials were combined in a beaker and mixed until dissolution, at
ambient temperature. The co-surfactant to surfactants (CoS/S)
weight ratio was preserved 1:5.
[0095] Carbomer aqueous solution was prepared by dispersing 0.05 g
of Carbopol 974NF in 10 g of double-distilled water and mixing at
room temperature until homogeneous mucilage was obtained, for about
5-10 minutes. Thereafter, sodium hydroxide solution was used to
adjust the pH, about 50 .mu.L, and the solution was stirred for
additional 10 minutes. The solution contained 0.5% wt of
polymer.
[0096] Microemulsion was prepared by mixing in a beaker 8.5 g of
the oil phase with 0.2 g of polymer solution and 0.8 g of water, at
room temperature. The water-in-oil microemulsion with 15% of
aqueous phase and 0.01% wt of polymer was spontaneously formed.
[0097] Crosslinking was performed at 35.degree. C. by adding 0.5 g
of calcium gluconate solution, 0.645% wt in water, into the
vigorously stirred microemulsion (300 rpm), using a peristaltic
pump operated at a controlled rate of 0.3 ml/min. The obtained
nanodispersion was stirred for additional 15 minutes.
[0098] The nanoparticles were separated from the microemulsion by
diluting the bulk with 10 mL of wash solution containing 6% wt of
sodium chloride and 0.5% wt of mannitol. The obtained mixture was
allowed to separate in a burette for 4 hours or left overnight, and
the bottom aqueous phase containing the nanoparticles was
removed.
Specific Examples 1.2-1.9--Calcium-Carbomer Nanoparticles,
Different Variables
[0099] Various nanoparticles were prepared based on the process of
the Example 1.1. The formulations are shown in the table 1 below.
IPP is isopropyl palmitate; S1 is glyceryl oleate; S1 is Labrasol;
Co-S is propylene carbonate; Carbomer is Carbopol 974NF; CG is
calcium gluconate aqueous solution; DDW is double-distilled water;
and AqC is aqueous content.
TABLE-US-00001 TABLE 1 Aqueous phase Oil Surfactants Carbomer CC,
AqC IPP, Co-S, 0.25% DDW, % wt. g S.sub.1, g S.sub.2, g g (w/w) , g
g (w/w) % 1.2 1.125 1.64 4.922 1.312 0.4 0.1 0.645 10 1.3 1.06 1.55
4.65 1.24 0.4 0.6 0.645 15 1.4 1 1.458 4.375 1.167 0.4 1.1 0.645 20
1.5 1 1.458 4.375 1.167 0.4 1.1 0.215 20 1.6 1 1.458 4.375 1.167
0.4 1.1 0.429 20 1.7 1 1.458 4.375 1.167 0.4 1.1 1.074 20 1.8 1
1.458 4.375 1.167 0.24 1.26 0.387 20 1.9 1 1.458 4.375 1.167 0.56
0.94 0.902 20
[0100] Formulations 1.2-1.4 demonstrate microemulsions with
different aqueous contents (10, 15, and 20% wt). Formulations
1.4-1.7 demonstrate microemulsions with 20% wt aqueous contents and
different Ca.sup.2+/Carbomer ratios (i.e. 1:10, 2:10, 3:10, 5:10).
Formulations 1.4, 1.8 and 1.9 demonstrate microemulsions with 20%
wt aqueous content, Ca.sup.+2/Carbomer ratio of 3:10 with different
polymer concentrations (i.e. 0.03, 0.05, 0.07% wt).
Specific Examples 1.10-1.19--Chloramphenicol-Loaded
Nanoparticles
[0101] Chloramphenicol-loaded nanoparticles were prepared
similarly. Specifically, the microemulsion was formed as in Example
1.1, and chloramphenicol, 0.5 g, was added into the microemulsion
to final concentration 5% wt, and stirred at 1200 rpm for 10
minutes. Crosslinking was performed as in Example 1.1. The
parameters are summarized in the Tables 2 and 3 below.
TABLE-US-00002 TABLE 2 1.10 1.11 1.12 1.13 1.14 C carbomer 0.2 wt %
0.1 wt % 0.067 wt % 0.05 wt % 0.04 wt % in water C water in 6.25%
12.5% 18.75% 25% 31.25% ME C carbomer 1.25 wt % 1.25 wt % 1.25 wt %
1.25 wt % 1.25 wt % in ME
TABLE-US-00003 TABLE 3 1.15 1.16 1.17 1.18 1.19 C Ca.sup.+2/ 0.215
wt % 0.43 wt % 0.645 wt % 1.29 wt % 2.15 wt % solution C Ca.sup.+2
20% 20% 20% 20% 20% solution/ ME C Ca.sup.+2/ 0.043 wt % 0.086 wt %
0.129 wt % 0.258 wt % 0.43 wt % ME
[0102] Formulations 1.10-1.14 demonstrate varying water content in
microemulsion. Formulations 1.15-1.19 demonstrate varying calcium
concentration in the microemulsion.
Specific Examples 1.20-1.29--Doxorubicin-Loaded Nanoparticles
[0103] Doxorubicin-loaded nanoparticles were prepared similarly.
Specifically, the microemulsion was formed as in Example 1.1, with
surfactants-to-oil ratio of 88:12; doxorubicin, 4 mg, as 0.8 mL of
aqueous solution with concentration 5 mg/mL, was added into the
aqueous polymer solution, to yield a final microemulsion
concentration of 400 .mu.g/mL, and stirred at 1200 rpm for 10
minutes, until clear solution was obtained. The pH was adjusted to
7.5 with about 40 .mu.L of 0.5% wt triethanolamine solution in
water. Thereafter the aqueous phase was added into the oil, as in
the Example 1.1, to form the microemulsion. Crosslinking was
performed as in Example 1.1, stirring at 1200 rpm and adding the
crosslinker solution using a syringe pump (NE 1000, New Era Pump
Systems, NY) at the same rate 0.3 mL/min.
[0104] Formulation parameters are summarized in the table 4 below.
C pol is concentration of the polymer in the microemulsion,
Ca.sup.+2/pol is the ratio between the crosslinking ion and the
polymer, XL T is the crosslinking temperature, AqC is aqueous
content in the microemulsion, DOX is doxorubicin concentration as
.mu.g/mL, and NP is the resulting nanoparticle size in
nanometers.
TABLE-US-00004 TABLE 4 C pol. Ca .sup.+2/ (%) pol. XL T AqC DOX NP
1.6 0.01 3:10 25.degree. C. 16 0 190.8 1.20 0.01 1:10 25.degree. C.
16 41.7 228.7 1.21 0.01 3:10 25.degree. C. 16 40.5 233.1 1.22 0.01
5:10 25.degree. C. 16 42.1 219.4 1.23 0.01 1:1 25.degree. C. 16
34.6 245.6 1.24 0.02 3:10 25.degree. C. 16 35.2 225.3 1.25 0.04
3:10 25.degree. C. 16 34.8 226.1 1.26 0.01 3:10 25.degree. C. 20
34.4 239.9 1.27 0.01 3:10 25.degree. C. 25 35.5 165.0 1.28 0.01
3:10 30.degree. C. 16 36.3 242.5 1.29 0.01 3:10 35.degree. C. 16
35.0 194.5
[0105] Formulations 1.20-1.23 demonstrate several calcium gluconate
concentrations in the microemulsion (16% w/w of total aqueous
phase, and 0.01% polymer concentration were kept constant) at the
following Ca.sup.++ ions/polymer ratios: 1:10, 3:10, 1:2, and 1:1.
Formulations 1.24-1.25 demonstrate additional polymer
concentrations, 0.02, and 0.04% wt, with the Ca.sup.++ ions/polymer
ratio of 3:10 and the microemulsion aqueous phase 16%. Formulations
1.26-1.27 demonstrate additional aqueous concentrations, 20% and
25%. Formulations 1.28-1.29 demonstrate composition 1.26 with
different crosslinking temperatures, 30.degree. C. and 35.degree.
C.
Specific Examples 1.30-1.37--Curcumin-Loaded Nanoparticles
[0106] Curcumin-loaded nanoparticles were prepared similarly to
doxorubicin nanoparticles. Specifically, the microemulsion was
formed as in Example 1.1 with surfactants-to-oil ratio of 90:10,
and curcumin, 200 mg, was added into the formed microemulsion to
final concentration 2.1% wt, and stirred at 500 rpm for 10 minutes,
until clear solution was obtained. No further pH adjustment was
performed. Crosslinking was performed as in Example 1.1, stirring
at 1200 rpm and adding the crosslinker solution using a syringe
pump (NE 1000, New Era Pump Systems, NY) at the same rate 0.3
mL/min. Temperature was controlled with a heat bath set to
35.degree. C.
[0107] The formulations 1.30-1.37 are based on 1.2-1.9, with
curcumin added as described and surfactants-to-oil ratio adapted to
90:10.
[0108] The specific formulations of the examples described can be
readily obtained from the specific features as exemplified
herein.
Example 2. Characterization of the Nanoparticles
2.1. Microscopic Observations
[0109] The Carbopol 974P NF polymer or carbomer homopolymer type B
(USP/NF; carboxypolymethylene) is a high molecular weight polymer
of acrylic acid cross-linked with allylpentaerythritol. Since it
contains 56%-68% of free carboxylic acid groups, it can be further
crosslinked by calcium ions at alkaline pH and precipitated.
Referring now to FIG. 2, morphology of NPs as observed under the
transmission electron microscope (TEM) and scanning electron
microscope (SEM) are demonstrated. Photographs (A) and (B) are
transmission electron microscopic (TEM) images of CHL-loaded
(magnification .times.8000) and unloaded NPs (magnification
.times.15000), respectively. Photograph (C) is a scanning electron
microscopic image of loaded NPs (magnification .times.200000).
According to TEM analysis (FIGS. 2A and 2B), unloaded NPs are
smaller (77-180 nm in diameter) than CHL-loaded NPs (200-40 nm in
diameter). The dependency of NPs size on drug-loading can be
explained by (a) increase of microemulsion nanodroplets in presence
of CHL, resulting in larger NPs after crosslinking, and/or (b)
decrease in the crosslinking process due to intercalation of the
drug between the polymer chains. Although TEM analysis is accurate
and reliable, it inspects only a limited subset of the entire
sample, which may theoretically be a non-representative sample. In
addition, the size difference between loaded and unloaded NPs may
be obtained due to the drying technique during sample preparation.
The unloaded nanoparticles may shrink during the drying process,
while shrinking of CHL-loaded NP may be limited by drug occupying
the pores and channels. In addition to the NP size, FIGS. 2A and 2B
have shown that the unloaded NPs adopted a spherical shape with a
smooth surface, while the loaded NPs were surrounded by a roughened
surface. To understand the character of this granular structure,
loaded NPs were further analyzed by SEM (FIG. 2C), which has
clearly demonstrated a roughened structure. Though this interesting
surface morphology is clearly related to drug incorporation within
the polymeric network, its structure is still remaining to be
investigated.
2.2. Influence of Calcium Ions Levels
[0110] The nanoparticle size as measured by DLS was first evaluated
in a series of five NP dispersions produced with various
concentrations of calcium gluconate. The microemulsion used for
these preparations contained 0.01% Carbopol and 30% aqueous phase.
Referring now to FIGS. 3A and 3B, exemplified is the effect of
Ca.sup.+2 gluconate level on average particle size (in diameter) of
CHL-loaded and unloaded NPs (A) and on CHL NP obtained in the NP
dispersion after salting out and separation from the microemulsion
(B). The aqueous phase in microemulsion was 30% and polymer
concentration was 0.01% (w/w). It can clearly be seen in FIG. 3A
that the average size of drug-loaded NPs was consistently larger
than that the size of unloaded NPs. The influence of drug uploading
on the NP size was in agreement with the abovementioned microscopic
observation. The results have also demonstrated that increase in
Ca.sup.+2 concentrations in the microemulsion resulted in increase
in NP size. With respect to the influence of drug uploading, it has
been shown (FIG. 3A) that NP size reached to maximum after addition
of 0.13% Ca.sup.+2 gluconate to CHL-containing microemulsion while
the maximal increase in the size of unloaded NPs required 0.26%
Ca.sup.+2 gluconate. Without being bound by a theory, yet in
accordance with the TEM observation previously discussed, this
phenomenon may indicate a decrease in the crosslinking reaction
capability due to intercalation of the drug between the polymer
chains, which may also result in size increase by surface
interaction and aggregation. The percent yield of drug loading in
the NP dispersion as a function of Ca.sup.+2 gluconate
concentrations is presented in FIG. 3B. As in the data regarding NP
size, CHL loading in NP dispersion increased by elevating Ca.sup.+2
levels and reached to maximum after addition of 0.13% Ca.sup.+2
gluconate to CHL-containing microemulsion. Further elevation of
Ca.sup.+2 caused to a gradual decrease in drug loading capacity,
which may indicate that reaction of the polymer with more Ca.sup.+2
resulted in displacement of the drug out of the NPs.
2.3. Influence of the Aqueous Phase Content in Microemulsions
[0111] The NP size (as measured by DLS) was also evaluated in a
series of five formulations containing various contents of aqueous
phase, specifically, along dilution line DL20. In this experiment,
the concentrations of the polymer and calcium gluconate were kept
constant at 0.01% and 0.086%, respectively.
[0112] Referring now to FIG. 4, the effect of aqueous phase content
of the microemulsion on the average size (in diameter) of
CHL-loaded and unloaded NPs is demonstrated. The concentrations of
the polymer and calcium gluconate were kept constant at 0.01% and
0.086%, respectively. Increase in NP size was effective only above
35% of water content (FIG. 4). Although the polymer and the
cross-linking agent contents were kept constant in the
microemulsion, their concentrations in the inner aqueous droplets
decreased with increasing water content. The low concentrations of
the reactants in the aqueous phase resulted in loose, swollen and
larger NPs. The aqueous nanodroplets can also increase in size in
microemulsions containing larger water phase, however, the size of
the droplets are around 10 nm in diameter and only incremental
changes occur in microemulsions containing more water. It is only
reasonable, therefore, that these small changes in the nano-droplet
size should not affect NPs whose size is between 200-300 nm.
[0113] Similarly to the results in FIG. 3A, FIG. 4 demonstrates the
significant effect of CHL on NP size.
[0114] Referring now to FIG. 5, the effect of aqueous phase content
of the microemulsion on CHL loaded in the NP dispersion produced by
0.086% and 0.129% Ca++ gluconate concentrations is exemplified.
Polymer concentration was 0.01% (w/w). FIG. 5 presents the drug
loading in the NP dispersions, which were produced by using two
levels of Ca.sup.+2 at various water concentrations in the
microemulsion precursor. As shown, more CHL was loaded in NPs of
formulations containing 0.13% calcium gluconate than NPs formed at
0.068% concentration, which is in good agreement with the data
shown in FIG. 3B. FIG. 5 also shows that in formulations containing
both Ca.sup.+2 concentrations the drug loading capacity of NPs
increased proportionally to the increase in water content. The
extent of the increase in CHL loading was more significant when the
aqueous phase was larger than 40% of the microemulsion. In light of
the data presented in FIGS. 4 and 5, it may be concluded therefore
that more drug can be entrapped in larger NPs than relatively
small-size NPs. It should be noted that because the polymer content
is kept constant in all formulations, dispersions of large NPs
containing less NP units per volume and less surface area for drug
diffusion than dispersions of small-size NPs. These parameters have
been estimated in the modeling section below.
[0115] In conclusion of the characterization of NPs produced by the
present process, NP dispersion formed after salting out of a
microemulsion template containing 25% aqueous phase, 0.01% Carbopol
polymer concentration, and 0.086% of calcium gluconate was found to
be optimum in regard to particle size (200-300 nm) and drug
loading.
Example 3. Drug Release and Modeling
3.1. Calculation of k' for Chloramphenicol
[0116] The apparent dialysis bag permeability constant (k') was
calculated according to the method described in `Materials and
Methods` section 2.6.
[0117] Referring now to FIGS. 6A and 6B, exemplified is (A) a plot
of In[C_1 0 V_1-C_2 (t)(V_2+V_1)] vs dialysis time of four
different concentrations of chloramphenicol in NaCl 0.9% w/v; and
(B) chloramphenicol recovery and the apparent permeability constant
(k') values obtained from dialysis of various chloramphenicol
concentrations in NaCl 0.9% w/v (V_1:donor solution,V_2:receiver
solution). The equilibrium between the donor and the receiver
solution was accomplished after about 4 hours for all samples. The
values of the apparent permeability constant (k') were determined
from the slope of the straight lines (R.sup.2=0.99) obtained from
the equation on top of FIG. 6A. As it shown in FIG. 6B, the
calculated value of the apparent permeability constant
(k'=1.207.+-.0.018 h.sup.-1) was negligibly affected by the initial
drug concentration. In addition to the k' determination, this
experiment examined the suitability of the dynamic dialysis
technique for drug release testing from NPs. Based on these
results, which have shown a total recovery of the drug in the
receiver medium for all samples (FIG. 6B), the dynamic dialysis
method was found as an appropriate technique for drug release
evaluation of CHL-NPs.
3.2. Chloramphenicol Release from NPs
[0118] As discussed previously, drug release from NPs may be
affected by the levels of drug loading. Therefore, two types of
differently drug-loaded NPs were tested in order to characterize
the mechanism. Both types of nanoparticles were prepared using
0.086% calcium gluconate and 0.01% Carbopol polymer, however, one
formulation (formulation 1.10) was containing 25% aqueous phase and
the other (formulation 1.14) 45% aqueous phase. Referring now to
FIG. 7, the results are shown therein. The formulation 1.10 with
25% of aqueous phase is represented by empty diamonds (.diamond.),
designated W1, whereas the 1.14 with 45% of aqueous phase is a
closed circle (o), designated W5.
[0119] Without being bound by a theory, the drug release of 1.10
may be attributed to matrix containing dissolved drug only,
indicating that the drug concentration in the particles is lower or
equal the solubility in the extraction medium (saline). The drug
release of 1.14 may be attributed to larger amounts of drug
entrapped in the nanoparticles, therefore the matrix may contain
the dispersed drug, i.e. the concentration of the drug in the
particle is above the solubility in the extraction medium. The
assumptions may be corroborated by suitable calculations.
Example 4. Production and Use of Nanoparticles Comprising Curcumin
or Doxorubicin
[0120] Spherical nanoparticles (NPs) loaded with curcumin and
doxorubicin have been prepared and characterized as disclosed
herein. The overall manufacturing process (microemulsion
preparation as a platform, polymer crosslinking, salting-out,
extrusion and lyophilization) was optimized, evaluated and
standardized for nanoparticle sizes (<200 nm), drug content,
entrapment efficiency and minimal passive release. In addition,
evaluation of the efficacy of anticancer drug loaded-NPs has begun
using MCF-7 cell line of breast cancer, indicating advantageous
activity of our product over anticancer drugs in plain
solutions.
4.1. Optimizing the Formulation Parameters of Curcumin
Nanoparticles
[0121] Several parameters were altered to characterize the formed
nanoparticles. The goal was to achieve a narrow range of size
distribution having diameters between 100-200 nm. The parameters
that were changed were the content of aqueous phase in the
microemulsion (ME) precursor, polymer quantity, and the
cross-linker concentrations. Referring now to FIGS. 8A-8C, the
dependence between the nanoparticle radius (R, given in nm) and the
aqueous content, designated "AqC" (8A), amount of polymer,
designated "Pol." (8B), and the amount of cross-linker, expressed
in molar percent, designated "Ca.sup.+2" (8C) is exemplified. FIG.
8A demonstrates formulations with different percentages of aqueous
phase content in ME (10%, 15%, 20% w/w) were prepared and their
nanoparticles size were analyzed. It should be noted that in this
series of formulations, the polymer quantity (but not its
concentrations in the aqueous phase) as well as the cross-linker
level were kept constant. The second set of formulations was
prepared with different quantities of the polymer. The results of
this experiment are shown in FIG. 8B. The mean particle radius is
shown versus the amount of polymer (Pol.), expressed in mg per
10-mL formulation. In the third set of experiments (FIG. 8C),
different levels of the cross-linker (0.0012, 0.0035, 0.0075,
0.0116, 0.0174 mol % of Ca.sup.+2 ions) were investigated. The
polymer content (10 mg %) and the aqueous phase percentage (25%)
were kept constant.
4.2. Nanoparticle Morphology--Curcumin
[0122] The shape of the nanoparticles was inspected by transmission
electron microscopy (TEM). Referring now to FIG. 9A and 9B, TEM
images of nanoparticles loaded with curcumin are exemplified.
4.3. Drug Loading--Curcumin
[0123] Referring now to FIG. 10, the reference curve obtained by
potentiometric titration of three different polymer amounts is
exemplified, from pH 4.5 to 9.0. The amount of polymer in the
solution, designated as "Pol.", is correlated with the amount of
sodium hydroxide 5 mM solution required for the titration. By this
assay the yield of the polymer in the process and its quantity in
the nanoparticles are determined. Table 5 below shows the results
of the drug loading tests.
TABLE-US-00005 TABLE 5 Analytical summary of curcumin loading on
NPs Calcium gluconate concentration (% w/v) 0.645% 1.0% X-linker
added (mole) 7.5E-06 11.6E-06 X-linker in pre-lyophilized 8E-07
(SD: .+-.1E-07) 1.16E-06 (SD: .+-.6E-08) product (mole) Polymer
conc. (mg/mL) 0.15 (SD: .+-.0.03) 0.15 (SD: .+-.0.03) Curcumin
concentration 34.47 (SD: .+-.0.15) 72.5 (SD: .+-.1.7) in NPs
(.mu.g/mL) Curcumin Loading 0.230 (SD: .+-.1E-03) 0.48 (SD:
.+-.0.01) (w [CUR]/w [Polymer])
4.4. Drug Release--Curcumin
[0124] The release behavior of curcumin from hydrogel nanoparticles
with drug loading of 0.48 w/w was determined at 37.degree. C. at pH
7 with in BSA (2% w/v) and NaCl (0.9% w/v) added to the dissolution
medium. The results are shown in the FIG. 11. The release test was
done on two different production batches and lasted about 48 hours.
It should be noted that the percentage of curcumin release from
nanoparticles was found to be between 6-7% during the first 24
hours, indicating that a second phase of slower release rate takes
place.
4.5. Drug Loading--Doxorubicin
[0125] Four batches of the doxorubicin-loaded product were produced
according described above. Previously, several attempts to load the
drug had revealed that pH adjustment was critical for NPs
formation. The salting-out technique was not changed; however, the
dissolution of DOX-HCl was carried out within the preparation of
the polymer solution (i.e., DOX was not introduced directly in
microemulsion), pH was adjusted and then microemulsion was
spontaneously formed by mixing with the oily phase. Particle size
was determined to be 100 nm in diameter.
4.6. Drug Release--Doxorubicin
[0126] The release of DOX from NPs was evaluated by incubating the
DOX-NP dispersion at 37.degree. C. in the donor compartment of a
Franz diffusion cell system (PermeGear, Inc., Hellertown, Pa.). The
diffusion area was 1.767 cm.sup.2 (15 mm diameter orifice), and the
receiver compartment volume was 12 mL. The solutions in the
water-jacketed cells were thermostated at 37.degree. C. and stirred
by externally driven, Teflon-coated magnetic bars. A synthetic
membrane (SnakeSkin Dialysis Tubing, 10,000 MW cutoff, 22 mm,
Thermo Fisher Scientific, Rockford, USA) was cut and placed on the
receiver chambers and the donor chambers were then clamped in
place. The receiver chamber was filled with phosphate buffered
saline (PBS, pH 7.4). Aliquots (0.5 mL each) of DOX-NP dispersion
or plain DOX solutions in water were applied over the membrane.
Samples (1 mL) were withdrawn from the receiver solution at
predetermined time intervals, and the receiver cell was replenished
up to its marked volume with fresh buffer solution each time.
Addition of PBS to the receiver compartment was performed with
great care to avoid trapping air beneath the membrane. The receiver
samples were taken into 1.5-mL vials and kept at -20.degree. C.
until analyzed by HPLC.
[0127] The release of doxorubicin was shown to be time dependent
over two days and even may be longer. The graphs of cumulative
release of doxorubicin are exemplified in the FIGS. 12A and
12B.
4.7. Cytotoxic Studies--MCF7 Cell Line
[0128] The direct effect of curcumin and doxorubicin on the
viability of cultured cells was assessed in vitro using the MCF-7
cells (cell line of breast cancer). The cells (1.times.10.sup.4)
were plated in 96- well plates and were allowed to attach for 24 h
at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere. Fresh cell
medium was then prepared with test compounds in appropriate
dilutions (equivalent to their concentrations as determined by
HPLC). Cell growth after 48 h was assayed by MTT assay
[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
Sigma-Aldrich] and was evaluated by measuring the absorbance at 540
nm after 3 h of incubation in a medium containing 0.5 mg/ml MTT
(Sunrise.TM. absorbance plate reader, TECAN). Referring now to
FIGS. 13A-13C, the figures demonstrate the current results,
indicating that NPs loaded with anticancer drugs are more
efficacious than the drugs in plain solutions. FIG. 13A shows that
DOX-loaded NPs are more cytotoxic than DOX in plain solution (24 h
incubation). FIG. 13B demonstrates the same phenomenon with
curcumin. FIG. 13C presents dose response behavior of DOX-loaded
and unloaded NPs vs. solutions at various concentrations after 24-h
and 48-h incubation. It could be noted that due to the slow release
manner of the drug from NPs, the effect of the NPs is expressed
only from minimum 4-5 g/ml, compared with the solution which is
highly cytotoxic at 1 g/ml.
4.8. Animal Study
[0129] Female C57BL/6 mice (5-6 weeks old, 14-18 g) were obtained
from Harlan Laboratories Ltd (Jerusalem, Israel). Mice were housed
under humidity- and temperature-controlled conditions, and the
light/dark cycle was set at 12-h intervals. The animal protocol was
reviewed and approved by the Ben Gurion University of the Negev
Institutional Committee for the Ethical Care and Use of Animals in
Research, which complies with the Israeli Animal Welfare Law.
[0130] B16F0 melanoma cells were maintained in RPMI 1640 medium
supplemented with 10% bovine serum and 200 .mu.M L-glutamine, 10
units/mL penicillin, and 10 .mu.g/mL streptomycin (Biological
Industries, Beth Ha'Emek, Israel). The cells were kept at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere.
[0131] The mice were inoculated with melanoma B16F0 cells
(1.times.10.sup.6) subcutaneously in the right flank of the mice.
After tumors had been visualized a week later, the mice were
weighed and measured for tumor size (244.+-.213 mm.sup.3), and were
randomly sorted into three groups. The groups were assigned for
untreated control animals, treated animals with DOX-HCl solution
(40 .mu.g/ml, 0.2 mL/animal), and treated animals with DOX-NP
dispersion (40 .mu.g/ml, 0.2 mL/animal) (n=12 mice/group, n=11 mice
in the control group). The dose of DOX administered to the
treatment groups was 0.5 mg/kg twice a week, by subcutaneous (s.c.)
injection 1-cm proximal to the tumor at the frontal side. B-16
melanoma is an aggressive skin tumor that is not typically treated
with doxorubicin, therefore s.c. administration of the anti-cancer
drug has been thought to be more effective than the i.p. injection
which is widely-used in xenograft tumor models. Tumor volumes and
mouse body weights were measured routinely before each treatment.
Measurement of tumor size was performed with a caliper in two
dimensions, and individual tumor volumes (V) were calculated by the
formula: V=[length.times.(width).sup.2].times..pi./6.
[0132] The statistical differences between the release data
obtained from the various formulations were analyzed employing the
two-way unweighted means analysis of variance (ANOVA) test. The
statistical differences between the therapeutic effects of
different treatments on tumor growth were determined by two-tailed
Student's test. The differences among groups were considered
significant when p values<0.05.
[0133] Referring to FIG. 14A, the tumor growth inhibition by the
chemotherapeutic drug in terms of volume change during the course
of treatment (18 days) is shown. A comparison between drug-induced
inhibition and untreated control is presented. It is readily seen
that most pronounced tumor growth inhibition was observed in mice
treated with DOX-NPs. Compared with 40 .mu.g/ml DOX-HCl solution
treatment twice a week, the same dosage of DOX-loaded NPs
dramatically reduced tumor growth (Student t-test, p<<0.05).
The significant inhibition of tumor growth by DOX-NPs demonstrates
the efficacy of NP encapsulation. Referring now to FIG. 14B,
mortality occurred in the untreated group at 7 days (14 days after
inoculation) and increased rapidly to 73% mortality (or 27%
survival) at day 18. In comparison to the untreated control mice,
treatment apparently increased animal survival. However, only one
mouse of the twelve mice died in the group which received DOX-NPs
after 14 days of treatment while three more died at day 18 (67%
survival). This was compared to the treatment with DOX-HCl
solution, which summed up with a relatively higher mortality and
less survived animals (58% survival).
Example 5. Nanoparticles Preparation--a General Protocol
[0134] The method for producing nano-sized particles according to
the present invention is schematically presented in the flow sheet
shown in FIG. 15.
[0135] While the present disclosure has been illustrated and
described with respect to a particular embodiment thereof, it
should be appreciated by those of ordinary skill in the art that
various modifications to this disclosure may be made without
departing from the spirit and scope of the present disclosure.
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