U.S. patent application number 11/998841 was filed with the patent office on 2008-09-04 for tumor environment-induced ligand-expressing nanocarrier system.
Invention is credited to You Han Bae, Eun Seung Lee, Kun Na.
Application Number | 20080213379 11/998841 |
Document ID | / |
Family ID | 34622874 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080213379 |
Kind Code |
A1 |
Bae; You Han ; et
al. |
September 4, 2008 |
Tumor environment-induced ligand-expressing nanocarrier system
Abstract
Drug delivery compositions for specific delivery of a drug to a
tumor are described. These compositions include a core for
sequestering the drug and a shell to which a ligand is attached for
delivery of a drug to target cells. Since normal cells may also be
targeted by the ligand, the compositions embed the ligand in the
shell until the localized conditions surrounding the tumor cause
the ligand to be displayed on the surface of the shell. One
composition exhibits shrinkage of the shell at tumor pH, whereas
another composition exhibits extension of the ligand at tumor pH.
Still another composition causes the ligand to be exhibited at an
elevated temperature.
Inventors: |
Bae; You Han; (Salt Lake
City, UT) ; Na; Kun; (Suwon, KR) ; Lee; Eun
Seung; (Suwon, KR) |
Correspondence
Address: |
ALAN J. HOWARTH
P.O. BOX 1909
SANDY
UT
84091-1909
US
|
Family ID: |
34622874 |
Appl. No.: |
11/998841 |
Filed: |
November 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10948078 |
Sep 22, 2004 |
7371781 |
|
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11998841 |
|
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60505227 |
Sep 22, 2003 |
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Current U.S.
Class: |
424/490 |
Current CPC
Class: |
A61K 9/1075
20130101 |
Class at
Publication: |
424/490 |
International
Class: |
A61K 9/14 20060101
A61K009/14 |
Claims
1. A drug delivery composition comprising (a) a mixed polymeric
micelle comprising an inner core and an outer shell, wherein the
mixed polymeric micelle comprises a mixture of (i) an amphiphilic
block copolymer comprising an AB-block copolymer comprising a
poly(ethylene glycol)-poly(L-histidine) block copolymer or a
poly(ethylene glycol)-poly(L-lactic acid) block copolymer or a
poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) block
copolymer, or an ABA-block copolymer comprising a poly(ethylene
glycol)-poly(L-lactic acid)-poly(ethylene glycol) block copolymer
or a poly(ethylene glycol)-poly(lactic acid-co-glycolic
acid)-poly(ethylene glycol) block copolymer, or a BAB-block
copolymer comprising a poly(L-lactic acid)-poly(ethylene
glycol)-poly(L-lactic acid) block copolymer or a poly(lactic
acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic
acid-co-glycolic acid) block copolymer, or an (AB).sub.n
alternating multiblock copolymer comprising a (poly(ethylene
glycol)-poly(L-lactic acid)).sub.n alternating multiblock copolymer
or a (poly(ethylene glycol)-poly(lactic acid-co-glycolic
acid)).sub.n alternating multiblock copolymer, and (ii) a
pH-sensitive block copolymer having a ligand covalently bonded
thereto, wherein the pH-sensitive block copolymer comprises an
extended configuration at less than about pH 7.0 and a globular
configuration at about pH 7.4 or greater, and wherein the
pH-sensitive block copolymer having a ligand covalently bonded
thereto comprises a ligand/AB-block copolymer comprising a
ligand-poly(L-histidine)-poly(L-lactic acid) block copolymer or a
ligand-poly(L-histidine)-poly(lactic acid-co-glycolic acid) block
copolymer, or a ligand/ABA-block copolymer comprising a
ligand-poly(L-histidine)-poly(ethylene glycol)-poly(L-histidine)
block copolymer, or a ligand/BAC-block copolymer comprising a
ligand-poly(ethylene glycol)-poly(L-histidine)-poly(L-lactic acid)
block copolymer or a ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(lactic acid-co-glycolic acid) block
copolymer, or a ligand/BABA-block copolymer comprising a
ligand-poly(ethylene glycol)-poly(L-histidine)-poly(ethylene
glycol)-poly(L-histidine) block copolymer, or a ligand/BABC-block
copolymer comprising a ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(ethylene glycol)-poly(L-lactic acid)
or a ligand-poly(ethylene glycol)-poly(L-histidine)-poly(ethylene
glycol)-poly(lactic acid-co-glycolic acid), or a ligand/block
copolymer comprising a ligand-poly(L-histidine)-poly(ethylene
glycol)-poly(L-lactic acid) block copolymer; and (b) a drug
disposed in the inner core.
2. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises a portion thereof that is
hydrophobic at about pH 7.4 or greater and hydrophilic at less than
about pH 7.0.
3. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the
ligand-poly(L-histidine)-poly(L-lactic acid) block copolymer.
4. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the
ligand-poly(L-histidine)-poly(lactic acid-co-glycolic acid) block
copolymer.
5. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the
ligand-poly(L-histidine)-poly(ethylene glycol)-poly(L-histidine)
block copolymer.
6. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(L-lactic acid) block copolymer.
7. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(lactic acid-co-glycolic acid) block
copolymer.
8. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(ethylene glycol)-poly(L-histidine)
block copolymer.
9. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the
ligand-poly(L-histidine)-poly(ethylene glycol)-poly(L-lactic acid)
block copolymer.
10. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(ethylene glycol)-poly(L-lactic acid)
block copolymer.
11. The drug delivery composition of claim 1 wherein the
pH-sensitive block copolymer comprises the ligand-poly(ethylene
glycol)-poly(L-histidine)-poly(ethylene glycol)-poly(lactic
acid-co-glycolic acid) block copolymer.
12. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(ethylene
glycol)-poly(L-lactic acid) block copolymer.
13. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(ethylene
glycol)-poly(lactic acid-co-glycolic acid) block copolymer.
14. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(ethylene
glycol)-poly(L-histidine) block copolymer.
15. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(ethylene
glycol)-poly(L-lactic acid)-poly(ethylene glycol) block
copolymer.
16. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(ethylene
glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)
block copolymer.
17. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(L-lactic
acid)-poly(ethylene glycol)-poly(L-lactic acid) block
copolymer.
18. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the poly(lactic acid-co-glycolic
acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)
block copolymer.
19. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the (poly(ethylene
glycol)-poly(L-lactic acid)).sub.n alternating multiblock
copolymer.
20. The drug delivery composition of claim 1 wherein the
amphiphilic copolymer comprises the (poly(ethylene
glycol)-poly(lactic acid-co-glycolic acid)).sub.n alternating
multiblock copolymer.
21. The drug delivery composition of claim 1 wherein the
amphiphilic block copolymer comprises the poly(ethylene
glycol-poly(L-lactic acid) block copolymer, and the pH-sensitive
block copolymer comprises the
ligand-poly(L-histidine)-poly(ethylene glycol)-poly(L-lactic acid)
block copolymer.
22. The drug delivery composition of claim 1 wherein the
amphiphilic block copolymer comprises the poly(ethylene
glycol)-poly(L-histidine) block copolymer, and the pH-sensitive
block copolymer comprises the
ligand-poly(L-histidine)-poly(ethylene glycol)-poly(L-lactic acid)
block copolymer.
23. The drug delivery composition of claim 1 wherein the ligand
comprises folate.
24. The drug delivery composition of claim 1 wherein the ligand
comprises biotin.
25. A drug delivery composition comprising (a) a mixed polymeric
micelle comprising an inner core and an outer shell, wherein the
mixed polymeric micelle comprises a mixture of (i) an amphiphilic
block copolymer and (ii) a temperature-sensitive block copolymer
having a ligand covalently bonded thereto, wherein the
temperature-sensitive block copolymer comprises an globular
configuration at 37.degree. C. and an extended configuration at
about 38.degree. C. to about 45.degree. C.; and (b) a drug disposed
in the inner core.
26. A drug delivery composition comprising (a) a mixed polymeric
micelle comprising an inner core and an outer shell, wherein the
mixed polymeric micelle comprises a mixture of (i) an amphiphilic
block copolymer and (ii) a temperature-sensitive block copolymer
having a ligand covalently bonded thereto, wherein the
temperature-sensitive block copolymer comprises an extended
configuration at 37.degree. C. and a globular configuration at
about 38.degree. C. to about 45.degree. C.; and (b) a drug disposed
in the inner core.
27. A drug delivery composition comprising (a) a mixed polymeric
micelle comprising an inner core and an outer shell, wherein the
mixed polymeric micelle comprises a mixture of (i) a first
amphiphilic block copolymer comprising a ligand covalently bonded
thereto and (ii) a second amphiphilic block copolymer comprising a
biodegradable, pH-sensitive polymer block that comprises a globular
configuration at less than about pH 7.0 and comprises an extended
configuration at pH 7.4 or greater; and (b) a drug disposed in the
inner core.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/948,078, filed Sep. 22, 2004, which is hereby incorporated
by reference in its entirety, which claims the benefit of U.S.
Provisional Application No. 60/505,227, which is hereby
incorporated in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] This invention relates to drug delivery. More particularly,
this invention relates to compositions and methods or delivering
drugs to tumor cells.
[0004] Cancer cells are often characterized by specific antigens or
over-expressed receptors on cell surfaces. Such receptors provide
uptake routes for nutrients and signals from the surrounding
environment, which nutrients and signals are essential for active
growth of the cells. Examples include receptors for folic acid,
vitamin B12, and transferrin. S. D. Weitmann et al., Distribution
of the folate receptor GP38 in normal and malignant cell lines and
tissues, 52 Cancer Res. 3396-3401 (1992); J. F. Ross et al.,
Differential regulation of folate receptor isoforms in normal and
malignant tissues in vivo and in established cell lines.
Physiologic and clinical implications, 73 Cancer 2432-2443 (1994);
J. Holm et al., High-affinity folate binding in human choroid
plexus. Characterization of radioligand binding, immunoreactivity,
molecular heterogeneity and hydrophobic domain of the binding
protein. 280 Biochem. J. 267-272 (1991).
[0005] Active internalization into cells of nutrients, signal
molecules, or nanoparticulates via various processes, such as
endocytosis and phagocytosis, is a natural consequence of evolution
for survival of individual cells, signal transduction pathways, and
cellular immune reactions. These processes have now been utilized
as an important delivery mode for macromolecular and/or nano-sized
carriers, which ferry anti-cancer drugs, antisense
oligonucleotides, genes, or proteins, to tumor cells to kill cells
and/or suppress aggressive cell growth. P. S. Low & R. J. Lee,
Folate-mediated tumor cell targeting of liposome-entrapped
doxorubicin in vitro, 1233 Biochem. Biophys. Acta 134-144 (1995);
D. Goren et al., Nuclear delivery of doxorubicin via
folate-targeted liposomes with bypass of multidrug-resistance
efflux pump, 6 Clinical Cancer Res. 1949-1957 (2000).
[0006] Ligand-receptor and antigen-antibody interactions are
important modes in active tumor targeting and are frequently
utilized for designing carrier systems containing cytostatic agents
that minimize undesirable side effects of the drugs. S. P. Vyas et
al., Ligand-receptor mediated drug delivery: an emerging paradigm
in cellular drug targeting, 18 Critical Review in Therapeutic Drug
Carrier System 1-76 (2001).
[0007] Before ligand-receptor interactions occur, the anticancer
drug carriers should (a) avoid active and massive removal processes
in the body, which remove foreign particulates (carriers), and (b)
minimize interactions with normal, healthy cells. One approach for
accomplishing these requirements is surface decoration of the
carriers with hydrophilic polymers, particularly with poly(ethylene
glycol) (PEG). Such surface decoration minimizes the adsorption of
plasma proteins that mediate particulate uptake (opsonization) by
the reticuloendothelium system (RES). This provides a certain
degree of stealth effect for particulate carriers, which provides
prolonged circulation in the blood stream. Prolonged circulation in
the blood stream, in turn, results in a higher probability of the
drug reaching tumor sites by the enhanced permeability and
retention (EPR) effect in the defective and leaky vasculature in
the tumors. M. J. Roberts et al., Chemistry for peptide and protein
PEGylation, 54 Adv. Drug Deliv. Rev. 459-476 (2002); G. Molineux,
Pegylation: engineering improved pharmaceuticals for enhanced
therapy, Cancer Treat Rev. Suppl A:13-16 (2002). The introduction
of a ligand to the ends of PEG chains immobilized to the carrier
surface is also employed in the design of anticancer carriers for
active targeting or internalization.
[0008] Receptor-mediated endocytosis has received major attention
in the field of drug/gene delivery in the past few decades. This
process could be exploited for designing compositions and methods
for delivery of site-specific bioactive agents, in particular, for
the delivery of anti-cancer agents, because anti-cancer agents have
disadvantageous properties, such as nonspecific toxicity, lack of
tumor selectivity, and development of multi-drug resistance in
various tumor cells. To overcome these problems and to increase the
therapeutic index of the drugs, various tumor targeting moieties,
such as vitamins, sugars, and antibodies, have been investigated.
These target moieties showed to some extent the potential for the
effective treatment of tumors. Recently, some kinds of vitamins and
sugars have been used as tumor-targeting moieties, because their
activities, which can internalize with macromolecules in tumor
cells, were confirmed by in vitro tests. However, the successful
tumor targeting of carriers with the moieties in vivo has rarely
been reported. A reason for this, although tumor targeting combined
with receptor-mediated endocytosis is effective, is the same
receptors are present on various normal cell surfaces, with various
degrees of expression. For instance, even though the receptor of
folic acid, a B vitamin, is known to be over-expressed on cancer
cells (e.g., ovarian, breast, lung, kidney, and brain tumors), it
is also expressed on normal cells because folic acid is an
essential nutrient. S. D. Garbis et al., Determination of folates
in human plasma using hydrophilic interaction chromatography-tandem
mass spectrometry, 73 Anal. Chem. 5358-5364 (2001). Actually, it
was reported that the folate binding protein (albumin) was greatly
accumulated in liver and kidney compared to controls (albumin
without folate) in tumor and non-tumor bearing mouse models, while
accumulation in tumor sites was slightly increased. The reason for
this observation is unknown, however, two possibilities include:
(i) the total amount of folate receptors expressed in liver and
kidney is more than that in tumors, even though the folate receptor
is over-expressed in tumors, and (ii) the likelihood of folate
contacting and binding to liver and kidney is higher than that of
contacting and binding to tumors. This reasoning is supported by a
report that suggested that liver and kidney are the main organs for
folate homeostasis. This means that folate molecules bonded to
macromolecules could not serve very well as tumor-targeting
moieties in vivo. In particular, in the case of a sugar moiety as a
targeting ligand, this phenomenon is extreme. The receptors for
sugar moieties such as glucose, galactose, and mannose are
distributed at each specific organ. Thus, tumor-site targeting
using a sugar moiety is very difficult, because their receptors are
also expressed at normal sites. For example, even if the hepatic
asialoglycoprotein receptor, found only on hepatocytes (about
500,000 receptor per cell), were a very useful receptor for liver
targeting, the targeting of liver tumors using this receptor has
been seldom or never reported because the receptor is distributed
on all hepatic cells (tumor and normal cells). K. A. Deal et al.,
Cellular distribution of 111In-LDTPA galactose BSA in normal and
Asialoglycoprotein receptor-deficient mouse liver, 25 Nuclear Med.
& Biology 379-385 (1998). Thus, active internalization via
receptor-mediated endocytosis is not specific in nature to tumor
cells and inevitably causes side effects once internalized into
normal cells. Therefore, to design a more effective delivery
system, the development of a carrier system, which can expose a
ligand to specific-site as tumor site, is necessary.
[0009] The extracellular pH (pH.sub.e) of normal tissues and blood
is kept constant at pH 7.4 and pH 7.5, respectively, and their
intracellular pH (pH.sub.i) is kept constant at pH 7.2. However, in
most tumors the pH gradient is reversed (pH.sub.i>pH.sub.e)
Particularly, pH.sub.e is more acidic in tumors than in normal
tissues. I. F. Tannock & D. Rotin, Acid pH in tumors and its
potential for therapeutic exploitation, 49 Cancer Res. 4373-4384
(1989); S. K. Hobbs et al., Regulation of transport pathways in
tumor vessels: Role of tumor type and microenvironment, 95 Proc.
Nat'l Acad. Sci. USA 4607-4612 (1998); M. Stubbs et al., Causes and
consequences of tumour acidity and implications for treatment, 6
Opinion 15-19 (2000). Although there is variation in in vivo
pH.sub.e measurements of human patients having various solid tumors
(e.g., adenocarcinoma, squamous cell carcinoma, soft tissue
sarcoma, and malignant melanoma) in readily accessible areas
(limbs, neck, or chest wall) using needle type microelectrodes, the
mean pH value is reported to be 7.06, with a full range of
5.66-7.78. K. Engin et al., Extracellular pH distribution in human
tumors, 11 Int. J. Hyperthermia 211-216 (1995). The variation is
dependent on tumor histology and tumor volume. Recent measurements
of pH.sub.e by noninvasive technology, such as .sup.19F, .sup.31P,
or .sup.1H probes for magnetic resonance spectroscopy in human
tumor xenografts in animals, further verified consistently low
pH.sub.e. R. van Sluis et al., In vivo imaging of extracellular pH
using .sup.1H MRSI, 41 Magn. Reson. Med. 743-750 (1999); A. S. E.
Ojugo et al., Measurement of the extracellular pH of solid tumours
in mice by magnetic resonance spectroscopy: a comparison of
exogenous .sup.19F and .sup.31P probes, 12 NMR Biomed. 495-504
(1999). All measurements of pH.sub.e of human and animal solid
tumors by either invasive or noninvasive methods showed that more
than 80% of all measured values falls below pH 7.2. This tumor pH
can be further lowered by hyperglycemia. D. B. Leeper et al., Human
tumor extracellular pH as a function of blood glucose
concentration, 28 Int. J. Radiat. Oncol. Biol. Phys. 935-943
(1994).
[0010] The high rate of glycolysis of tumor cells under either
aerobic or anaerobic condition has been thought to be a major cause
of low pH.sub.e. The tumor cells synthesize ATP by both
mitochondrial oxidative phosphorylation and glycolysis.
[0011] Glycolysis produces two moles of lactic acids (pK.sub.a=3.9)
and two moles of ATP by consuming one mole of glucose. Hydrolysis
of ATP also produces protons. Despite the high production rate of
protons in tumor cells, their cytosolic pH, particularly of
resistant cells, remains alkaline, which is favorable for
glycolysis, by exporting protons out of the cells by unknown
mechanisms. This leads to low pH.sub.e with the help of inadequate
blood supply, poor lymphatic drainage, and high interstitial
pressure in tumor tissues. I. F. Tannock & D. Rotin, supra; S.
K. Hobbs et al., supra; M. Stubbs et al., supra. However, it is of
interest to note that glycolysis-deficient variant cells (lacking
lactate dehydrogenase) produced negligible quantities of lactic
acid but still presented an acidic environment. M. Yamagata et al.,
The contribution of lactic acid to acidification of tumours:
studies of variant cells lacking lactate dehydrogenase, 77 Br. J.
Cancer 1726-1731 (1998). This finding has been interpreted as
meaning that acidity is a phenotype of tumor cells rather than a
consequence of cell metabolic events. The acidic environment may
benefit the cancer cells, because it promotes invasiveness
(metastasis) by destroying the extracellular matrix of surrounding
normal tissues.
[0012] This tumor pH.sub.e can be further manipulated to shift to
even lower pH by administration of glucose. The rate of glucose
uptake by tumor cells is higher than that of normal cell due to
higher metabolism and greater production and secretion of lactic
acid. This can lead to lower pH of the extracellular matrix and can
be beneficial for the approaches described in this application.
[0013] In view of the foregoing, it will be appreciated that
providing a tumor-selective targeting system for drug delivery
would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
[0014] A feature of an illustrative embodiment of the invention is
to provide a tumor-selective targeting system for drug delivery
wherein internalization of the drug occurs-only in tumor cells and
not in normal cells.
[0015] A feature of another illustrative embodiment of the
invention is to provide a tumor-targeting drug delivery system
wherein an interacting ligand is buried in the surface hydrophilic
shell of core-shell type polymeric micelles, thus minimizing the
interactions with normal cells or tissues during circulation while
the ligands are exposed on the surface of the outer shell of the
carriers only when the carriers arrive at the tumor sites.
[0016] Still another feature of an illustrative embodiment of the
present invention is to provide a tumor-targeting drug delivery
system that interacts with tumor cells and causes receptor-mediated
endocytosis.
[0017] Yet another feature of an illustrative embodiment of the
invention is to provide polymeric nano-carriers that can express
the interacting ligand by lowering pH or increasing temperature
from normal blood to tumor site.
[0018] These and other features and advantages are addressed by
providing a drug delivery composition comprising (a) a mixed
polymeric micelle comprising an inner core and an outer shell,
wherein the mixed polymeric micelle comprises a mixture of (i) a
first amphiphilic block copolymer comprising a ligand covalently
bonded thereto and (ii) a second amphiphilic block copolymer
comprising a biodegradable, pH-sensitive polymer block that
comprises a globular configuration at less than about pH 7.0 and
comprises an extended configuration at pH 7.4 or greater; and (b) a
drug disposed in the inner core.
[0019] Another illustrative embodiment of the invention comprises a
drug delivery composition comprising (a) a mixed polymeric micelle
comprising an inner core and an outer shell, wherein the mixed
polymeric micelle comprises a mixture of (i) an amphiphilic block
copolymer and (ii) a pH-sensitive block copolymer having a ligand
covalently bonded thereto, wherein the pH-sensitive block copolymer
comprises an extended configuration at less than about pH 7.0 and a
globular configuration at about pH 7.4 or greater; and (b) a drug
disposed in the inner core.
[0020] Still another illustrative embodiment of the invention
comprises a drug delivery composition comprising (a) a mixed
polymeric micelle comprising an inner core and an outer shell,
wherein the mixed polymeric micelle comprises a mixture of (i) an
amphiphilic block copolymer and (ii) a temperature-sensitive block
copolymer having a ligand covalently bonded thereto, wherein the
temperature-sensitive block copolymer comprises an globular
configuration at 37.degree. C. and an extended configuration at
about 38.degree. C. to about 45.degree. C.; and (b) a drug disposed
in the inner core.
[0021] Yet another illustrative embodiment of the invention
comprises a drug delivery composition comprising (a) a mixed
polymeric micelle comprising an inner core and an outer shell,
wherein the mixed polymeric micelle comprises a mixture of (i) an
amphiphilic block copolymer and (ii) a temperature-sensitive block
copolymer having a ligand covalently bonded thereto, wherein the
temperature-sensitive block copolymer comprises an extended
configuration at 37.degree. C. and a globular configuration at
about 38.degree. C. to about 45.degree. C.; and (b) a drug disposed
in the inner core.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0022] FIGS. 1A-B show a schematic representation of shrinking of
the outer shell of the drug delivery micelle system, according to
an illustrative embodiment of the present invention, to expose the
tumor-targeting ligand at tumor pH.
[0023] FIGS. 2A-B show a schematic representation of extension of
the tumor-targeting ligand in the outer shell of the drug delivery
micelle system, according to an illustrative embodiment of the
present invention, to effect exposure of the ligand at tumor
pH.
[0024] FIG. 3A shows a synthetic scheme for folate amination. FIG.
3B shows a synthetic scheme for L-histidine blocking. FIG. 3C shows
a synthetic scheme for L-histidine ring-opening polymerization
initiated by aminated folate. FIG. 3D shows a synthetic scheme for
PLLA/PEG/polyHis-folate block.
[0025] FIG. 4A shows cell viability as a function of adriamycin
(ADR) concentration when delivered with Carrier 3 at pH 6.8 ( ) and
at pH 7.4 (.box-solid.).
[0026] FIG. 4B shows cell viability as a function of ADR
concentration when delivered with Carrier 1 at pH 7.4 ( ) and at pH
6.8 (.box-solid.) and with Carrier 2 at pH 7.4 (.tangle-solidup.)
and at pH 6.8 ().
[0027] FIG. 5A shows a synthetic scheme for biotinamination.
[0028] FIG. 5B shows a scheme for synthesis of biotin-polyHis.
[0029] FIG. 6 shows changes in turbidity of Carrier 1 ( ) and
Carrier 4 (.box-solid.) on decreasing pH; each polymeric micelle
sample (1.0 g/L) was mixed with avidin (0.1 g/L); transmittance of
each polymeric micelle sample was measured at 24 hours and
determined at 500 nm and was baseline-corrected with 0.1 g/L avidin
at each pH (n=3).
[0030] FIG. 7 shows flow cytometry analysis with Carrier 4 (5
.mu.g/mL) exposed to MCf-7/ADR.sup.R cells (1.times.10.sup.5 cells)
for 30 min at pH 7.2 and 7.0.
[0031] FIGS. 8A-L show confocal microscopy analysis of biotinylated
Carrier 4 mixed micelles (ADR-loaded at a concentration of 0.5
.mu.g/mL) with conjugation of FITC, exposed to MCf-7/ADR.sup.R
cells (1.times.10.sup.5 cells) for 30 min. FIGS. 8A-C show
fluorescence at pH 7.4 of FITC, ADR, and both FITC and ADR,
respectively. FIGS. 8D-F show fluorescence at pH 7.2 of FITC, ADR,
and both FITC and ADR, respectively. FIGS. 8G-I show fluorescence
at pH 7.0 of FITC, ADR, and both FITC and ADR, respectively. FIGS.
8J-L show fluorescence at pH 6.8 of FITC, ADR, and both FITC and
ADR, respectively.
[0032] FIGS. 9A-L show confocal microscopy analysis of biotinylated
Carrier 5 mixed micelles (ADR-loaded at a concentration of 1
.mu.g/mL) with conjugation of FITC, exposed to MCf-7/ADR.sup.R
cells (1.times.10.sup.5 cells) for 30 min. FIGS. 9A-C show
fluorescence at pH 7.4 of FITC, ADR, and both FITC and ADR,
respectively. FIGS. 9D-F show fluorescence at pH 7.2 of FITC, ADR,
and both FITC and ADR, respectively. FIGS. 9G-I show fluorescence
at pH 7.0 of FITC, ADR, and both FITC and ADR, respectively. FIGS.
9J-L show fluorescence at pH 6.8 of FITC, ADR, and both FITC and
ADR, respectively.
[0033] FIG. 10 shows cell viability of ADR-loaded Carrier 4 against
MCf-7/ADR.sup.R cells at pH 7.4 ( ), pH 7.2 (.box-solid.), pH 7.0
(.tangle-solidup.), and pH 6.8 ().
DETAILED DESCRIPTION
[0034] Before the present tumor-targeting drug deliver compositions
and methods are disclosed and described, it is to be understood
that this invention is not limited to the particular
configurations, process steps, and materials disclosed herein as
such configurations, process steps, and materials may vary
somewhat. It is also to be understood that the terminology employed
herein is used for the purpose of describing particular embodiments
only and is not intended to be limiting since the scope of the
present invention will be limited only by the appended claims and
equivalents thereof.
[0035] The publications and other reference materials referred to
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0036] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to a mixed polymeric micelle
containing "a drug" includes a mixture of two or more drugs,
reference to "an amphiphilic copolymer" includes reference to one
or more of such amphiphilic copolymers, and reference to "a ligand"
includes reference to two or more of such ligands.
[0037] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0038] As used herein, "comprising," "including," "containing,"
"characterized by," and grammatical equivalents thereof are
inclusive or open-ended terms that do not exclude additional,
unrecited elements or method steps. "Comprising" is to be
interpreted as including the more restrictive terms "consisting of"
and "consisting essentially of."
[0039] As used herein, "consisting of" and grammatical equivalents
thereof exclude any element, step, or ingredient not specified in
the claim.
[0040] As used herein, "consisting essentially of" and grammatical
equivalents thereof limit the scope of a claim to the specified
materials or steps and those that do not materially affect the
basic and novel characteristic or characteristics of the claimed
invention.
[0041] As used herein, "FITC" means fluorescein isothiocyanate, and
"ADR" or "DOX" means adriamycin or doxorubicin.
[0042] To reduce or minimize undesired interactions or undesired
uptake into normal sites, a biodegradable nanocarrier has been
developed wherein access to the ligand is controlled by lowering pH
and increasing temperature. In this embodiment, the ligand is
hidden in a hydrophilic shell of a core-shell nanostructure during
circulation in the blood. The hidden or shielded ligand is exposed
on the surface of the nanocarrier for interaction with tumor cells
and internalization in tumor cells once the carriers reach the
tumor sites via extravasation through leaky tumor vessels. The
nanoparticle comprises (a) an amphiphilic block copolymer without a
ligand, and (b) another amphiphilic block copolymer with a ligand
attached to a tumor-pH-recognizing polymer. This nanoparticle was
designed and prepared for targeting a pH as close to 7.2 as
possible, so that the carrier can be used for targeting a broad
range of solid tumors. The nanoparticle is configured for
distinguishing differences in pH as small as 0.2 pH units.
[0043] Ligands or targeting moieties that can be used according to
the present invention include all types of ligands that are
internalized via receptor-mediated endocytosis. Categories of such
ligands include vitamins, monoclonal antibodies, tumor necrosis
factor-related apoptosis including ligand (TRAIL), endogenous
ligands, immunological ligands, glycoconjugates, organ specific
ligands, and the like.
[0044] Illustrative vitamins according to the present invention
include folate, biotin, retinoic acid, thiamine, niacin,
riboflavin, pantothenic acid, pyroxidine, and the like.
[0045] Illustrative monoclonal antibodies according to the present
invention include monoclonal antibodies specific to Fc, Fab', or
complement; anti-idiotype or anti-anti-idiotype monoclonal
antibodies against tumor epitopes; bispecific antibodies and
antibody-enzyme conjugates; haptens; Fab' or F(ab).sub.2 fragments;
single-chain Fv fragments (scFv); immunotoxins and chimeric toxins;
Fab' fragments of the monoclonal antibody Mu-9 (colon-specific
antigen p(CSAp)); PK4S sheep anti-CEA (carcino embryonic antigen);
human milk fat globule 1 (HMFG1; polymorphic epithelial mucin (PEM,
epithelial ovarian tumor)); OC 125 (tumor specific cell-surface
antigen CA-125, ovarian cancer; tumor vascular endothelium
monoclonal antibody (TES-23)); KMT-17, fibrosarcoma; A2B5, CD09,
and IC.sub.2 (monoclonal antibodies that specifically bind to beta
cells in the pancreas).
[0046] Illustrative endogenous ligands that can be used according
to the present invention include transferrin, folate, lipoprotein,
epidermal growth factor, nerve growth factor, insulin,
.alpha.-fetoprotein, macroglobulin, and the like.
[0047] Illustrative immunological ligands that can be used
according to the present invention include recombinant CD
molecules, CD4/rCD4-toxin conjugate, major histocompatibility
complex (MHC) peptides, interleukins, interferons, ECM ligands
including RGD and ISGR, synthetic sLe.sup.x or SLE.sup.a analogs,
recombinant gp120 and gp41, and the like.
[0048] Illustrative glycoconjugates that can be used according to
the present invention include lectins, glycolipids,
asialoglycoprotein, neoglycoprotein, glycosides, viroproteins,
polysaccharides, lipopolysaccharides, glucose or derivatives
thereof, galactose or derivatives thereof, maltose or derivatives
thereof, mannose or derivatives thereof, and the like.
[0049] Illustrative organ-specific ligands that can be used
according to the present invention include liver-targeting
moieties, such as N-acetylgalactoseamine, .beta.-galactose,
.alpha.-galactose, lactose, glucose, mannose, fibronectin,
.alpha.2-macroglobulin, Fc (immune complexes, opsonized particle),
Cb3 (complement factors), fucose, insulin (insulin receptor),
epidermal growth factor, IgA, transferrin, low density lipoprotein
(LDL), high density lipoprotein (HDL), glycyrrhizin, glucose
residues, lactoseaminated ferritin (Lac-Fer), lactobionic acid,
biotin, negatively charged proteins, sulfated polysaccharides
(e.g., chondroitin sulfate, heparin), and the like; lung-targeting
moieties, such as antagonist G; pancreas-targeting moieties, such
as sulfonylurea, GLP-1, somatostatin, glutamic acid decarboxylase,
carboxypeptidase H, GM2-1 pancreatic islet ganglioside, ICA 69
autoantigen, imogen 38, and the like; estrogen receptor positive
tumors, such as cyclopentadienyl tricarbonyl metal complex and the
like.
[0050] Drugs that can be delivered using the drug delivery
compositions of the present invention include adriamycin or
doxorubicin, paclitaxel, 5-fluorouracil (5-FU), vinblastine
sulfate, daunorubicin, mitoxantrone, idarubicin, etoposide,
teniposide, vincristine, vinblastine, dactinomycin, valinomycin,
puromycin, mithramycin, colchicine, mitomycin-C, trimetrexate,
actinomycin-D, cyclophosphamide, mechlorethamine, ifosphamide,
camustine (BCNU), semustine (CCNU), cytarabine (Ara-C),
methotrexate, cisplatin, carboplatin, bleomycin, hydroxyurea, and
the like.
[0051] Other bioactive agents that can be delivered according to
the present invention include proteins, such as interferon-.alpha.
(IFN-.alpha.), interferon-.gamma. (IFN-.gamma.), aldesleukin
(IL-2), atriopeptin III, cyclosporin A, insulin, ovalbumin, LHRH,
somatostatin, thyrotropin, t-PA, sargramostim (CSF-GM),
antithrombin III, calcitonin, erythropoietin, and the like;
peptides, such as growth factor, GLP-1, and the like; nucleic
acids, such as nucleic acids that encode therapeutic agents,
antisense agents, and the like; toxins; and the like.
[0052] Numerous polymers can be used according to the present
invention for making of pH-sensitive micelles. For example,
amphiphilic polymers that can be used according to the present
invention include amphiphilic polymers, such as poly(ethylene
oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock
copolymers, such as the PLURONIC.TM. block copolymers.
[0053] Biodegradable polymer blocks that can be used for making
copolymers include poly (L-lactic acid) (PLLA),
poly(lactide-co-glycolide) (PGLA), poly(glycolic acid) (PGA),
polyorthoester, polyanhydride, poly(histidine) (polyHis) and
polyHis copolymers.
[0054] Non-biodegradable polymer blocks that can be used for making
copolymers include poly(alkylcyanoacrylate),
poly(ethylcyanoacrylate), poly(isohexylcyanoacrylate), and
derivatives thereof.
[0055] Further, pH-sensitive polymers that can be used for
attachment to a ligand include poly(histidine) (poly(His)) and
poly(His) copolymers. Comonomers that can be used with L-histidine
for making poly(His) copolymers include leucine, alanine,
phenylalanine, and the like.
[0056] Still further, pH-sensitive polymers that can be used in the
outer shell include polymers containing poly(sulfonamide) groups
and copolymers thereof, and poly(His) and poly(His) copolymers.
Comonomers that can be used with L-histidine for making poly(His)
copolymers include leucine, alanine, phenylalanine, and the
like.
[0057] Temperature sensitive polymers for use in the outer shell
for making temperature sensitive micelles include
poly(N-isopropylacrylamide) and its copolymers, and PEG/PLLA
triblock or multiblock copolymers.
[0058] Poly(His) and its copolymers should generally be used in the
molecular weight range of about 300 to about 80,000. Such polymers
in the range of about 600 to about 10,000 are illustrative. PEG
blocks should generally be used in the molecular weight range of
about 300 to about 20,000. Such polymers in the range of about 1000
to about 10,000 are illustrative. The total molecular weight of
block copolymers used according to the present invention should
generally be in the molecular weight range of about 600 to about
100,000; and illustratively in the range of about 2000 to about
20,000.
[0059] Types of block copolymers that are used as pH-sensitive
polymers with a ligand attached thereto include ligand/AB-type
block copolymers, such as ligand-poly(His)-PLLA and
ligand-poly(His)-PLGA; ligand/ABA-type block copolymers, such as
ligand-poly(His)-PEG-poly (His); ligand/BAC-type block copolymers,
such as ligand-PEG-poly(His)-PLLA and ligand-PEG-poly(His)-PLGA;
ligand/BABA-type block copolymers, such as
ligand-PEG-poly(His)-PEG-poly(His); ligand/BABC-type block
copolymers, such as ligand-PEG-poly(His)-PEG-PLLA and
ligand-PEG-poly(His)-PEG-PGLA; and the like.
[0060] Block copolymers that can be used in the outer shell of the
pH-sensitive micelles include AB-type block copolymers, such as
PEG-poly(His), PEG-PLLA, PEG-PLGA, poly(sulfonamide)-PLLA, and
poly(sulfonamide)-PLGA; ABC-type block copolymers, such as
PEG-poly(sulfonamide)-PLLA and PEG-poly(sulfonamide)-PLGA; ABA-type
block copolymers, such as PEG-PLLA-PEG; BAB-type block copolymers,
such as PLLA-PEG-PLLA; (AB).sub.n-type alternating multiblock
copolymers, such as (poly(sulfonamide)-PEG).sub.n random multiblock
copolymers; and the like.
[0061] A first illustrative embodiment of the invention is a
ligand-exposing system comprising an outer shell that if configured
for shrinking at tumor pH (e.g., using a sulfonamide-based
biodegradable polymer). This micelle system has a ligand that can
be exposed by the shrinking of pH-sensitive outer shell at tumor
pH. FIGS. 1A-B show a schematic representation of a portion of a
micelle comprising a pH-sensitive outer shell made up of a
pH-sensitive polymer 10 and a ligand-bearing polymer 12. At pH
7.4-7.5, such as is encountered in normal tissues and the
bloodstream, the pH-sensitive polymer 10 is in an extended
configuration such that is shields or hides the ligand 14 (FIG.
1A). At tumor pH, the pH-sensitive polymer 10 contracts or shrinks,
thus exposing the ligand 14 on the surface of the micelle and
leaving the ligand 14 available for interaction with cell surface
receptors and the like. Thus, the ligand in this system cannot
interact to its receptor at normal pH because it is covered with
the hydrophilic outer shell. However, at tumor pH it can easily
interact and internalize via endocytosis due to the outer shell
shrinking or contracting.
[0062] For example, this system can be prepared by the mixing of
two different amphiphilic block copolymers: one is ligand/PEG
(3.4K)/PLLA (3K), the other is PEG (2K)/Biodegradable pH-sensitive
polymer (3K)/PEG (2K)/PLLA (3K). The development of biodegradable
pH-sensitive polymer responsive to tumor pH is a rate-determining
step in this system because extracellular tumor pH (<7.0) is
slightly lower than the normal pH (7.4). To prepare an outer shell
that shrinks or contracts at tumor pH, a sulfonamide group is used.
"Sulfonamide" is used herein to refer to derivatives of
para-aminobenzene sulfonamide (sulfanilamide). It is a weak acid
because the hydrogen atom of the amide nitrogen (N.sup.1) can be
readily ionized to liberate a proton in solution. By introducing a
selected sulfonamide group into water soluble polymers, a new class
of pH sensitive polymers has been synthesized. These polymers
demonstrate a first-order-like transition in solubility and
swelling by pH. Selecting an appropriate sulfonamide or changing
the polymer composition tailors this transition pH to selected
conditions. For examples, a selected, representative sulfonamide of
4-amino-N-[4,6-dimethyl-2-pyrimidinyl]benzene sulfonamide
(sulfamethazine; CAS No. 57-68-1) with its known pK.sub.a of 7.4,
was modified with methacryloyl chloride to produce a polymerizable
sulfonamide monomer. Unlike conventional pH-sensitive polymers,
this copolymer of the sulfonamide monomer and
N,N-dimethylacrylamide demonstrated a reversible first-order like
transition in solubility by pH. By varying the type of sulfonamide
and the copolymer composition, a broad range of the transition pH
can be targeted. Also, to utilize the pH-responsive property of
sulfonamides in targeting tumor pH.sub.e, a new class of
pH-responsive polymers was synthesized by conjugating a
sulfonamide, sulfadimethoxine (SDM; CAS No. 122-11-2), to
succinylated pullulan acetate (PA). The polymers formed
self-assembled PA/SDM hydrogel nanoparticles in aqueous media,
which was confirmed by fluorometry and field emission-scanning
electron microscopy. The nanoparticles showed good stability at pH
7.4, but shrank and aggregated below pH 7.0 due to the
sulfonamide's property of switching of from hydrophilic to
hydrophobic. The DOX release rate from the PA/SDM nanoparticles was
pH-dependent and significantly enhanced below pH 6.8. Furthermore,
a sulfonamide homopolymer was synthesized by radical polymerization
of a vinylated SD (SD monomer; SDM) using 2-aminoethanethiol as a
chain transfer agent to enhance the pH-sensitivity. For the
preparation of pH-sensitive polymeric micelle, the polymer was
coupled to a carboxylic PEG (Mw=2,000)/PLLA (Mw=3,500) block
copolymer. The micelle showed an aggregation behavior below pH 7.0
due to the presence of deionized SDM on the surface, while the
other micelles were stable. The polymeric micelle sharply responded
to the change in pH around the physiological pH.
[0063] An illustrative variation of this embodiment of the
invention comprises a biodegradable polymer based on sulfonamide.
To create a biodegradable sulfonamide polymer at a middle position
of a block copolymer, a poly(amino acid-grafted-sulfonamide) is
synthesized and then conjugated to aminated PEG. The resulting
PEG/poly(amino acid-grafted-sulfonamide) copolymer is then coupled
to carboxylic PEG/PLLA block copolymer to result in PEG/poly(amino
acid-grafted-sulfonamide)/PEG/PLLA. Micelles that expose the ligand
at tumor pH are then prepared by the mixing of PEG/poly(amino
acid-grafted-sulfonamide)/PEG/PLLA and ligand/PEG/PLLA block
copolymer.
[0064] A second illustrative embodiment of the invention comprises
a ligand expansion system, wherein the ligand is shielded or hidden
at normal pH, but exposed at tumor pH. This embodiment comprises a
mixed micelle comprising (a) a pH-sensitive block copolymer with an
attached ligand, and (b) an amphiphilic block copolymer. FIGS. 2A-B
a schematic representation of a portion of a micelle comprising a
pH-sensitive outer shell. The outer shell comprises a pH-sensitive,
ligand-bearing polymer 20 and a second polymer 22. At normal pH,
the ligand 24 is shielded or hidden by the second polymer 22 of the
outer shell (FIG. 2A), because the ligand-bearing polymer 20 is in
a compact configuration. At tumor pH, however, the ligand-bearing
polymer 20 is in an extended or expanded configuration (FIG. 2B),
thus exposing the ligand such that it can interact with receptors
and the like on the cell surface of tumors. Thus, even though the
ligand will be hidden at normal pH, it will be accessible at
extracellular tumor pH, because the pH-sensitive portion in the
polymer can be switched from hydrophobic (at normal pH) to
hydrophilic one (at extracellular tumor pH). To endow
pH-sensitivity to the ligand-bearing polymer, a poly(L-histidine)
block is used. L-Histidine is a basic amino acid with a pK of 6.5.
This pK value is influenced by the polymerization of histidine,
comonomer, and microenvironment. The imidazole ring in L-histidine
has a lone pair of electrons on the unsaturated nitrogen, which
endows an amphoteric nature to poly(L-histidine) (polyHis). PolyHis
is protonated at pH 5.0-7.0, being influenced by the molecular
weight of poly(L-histidine). PolyHis with a molecular weight of
>10,000 was reported to be soluble only below pH 6.0 by
protonation.
[0065] Poly(L-histidine) was synthesized using an aminated ligand
as an initiator. The polymer was coupled to PEG/PLLA block
copolymer. The extended ligand system is easily made by mixing of
PEG/PLLA and ligand/poly(L-histidine)/PEG/PLLA block
copolymers.
EXAMPLE 1
[0066] To demonstrate the invention, two vitamins (folate and
biotin) were selected as ligands, and several micelle systems were
prepared, as described below.
[0067] Carrier 1 comprised poly(His5K)/PEG2K block copolymer
and
[0068] PLLA3K/PEG2K (60:40 wt %) as a mixed micelle control. Such
micelles had an average diameter of about 100 nm.
[0069] Carrier 2 comprised poly(His5K)/PEG2K and
PLLA3K/PEG2K-folate (60:40 wt %) as a mixed micelle. Such micelles
also had an average diameter of about 100 nm.
[0070] Carrier 3 comprised poly(His5K)/PEG2K and
PLLA3K/PEG2K-poly(His1K)-folate (60:40 wt %) as a mixed micelle.
Such micelles also had an average diameter of about 100 nm.
[0071] Carrier 4 comprised poly(His5K)/PEG2K and
PLLA3K/PEG2K-poly(His1K)-biotin (60:40 wt %) as a mixed micelle.
Such micelles also had an average diameter of about 100 nm.
[0072] Carrier 5 comprised PLLA3K/PEG2K and
PLLA3K/PEG2K-poly(His1K)-biotin.
Synthesis of folate-poly(L-histidine)-PEG-PLLA
[0073] Histidine-NCA was synthesized using thionyl chloride after
the amine protection steps with benzylchoroformate and
2,4-nitrofluorobenzene, according to the procedure of U.S. patent
application Ser. No. 10/846,487, and E. S. Lee et al., Polymeric
micelle for tumor pH and folate mediated targeting, 91 J. Control.
Release 103-113 (2003), both of which are hereby incorporated by
reference. To synthesize PLLA-PEG-poly(L-histidine)-folate, amine
protected histidine-NCA (30 mmol; FIG. 3B) was dissolved in DMF (30
mL) in the presence of aminated folate (1 mmol; FIG. 3A) as an
initiator and reacted for 3 days at room temperature. After
reaction, diethyl ether was added to the solution for precipitation
of reactant. The preactivated PLLA-PEG-COOH with NHS was added in
same mole ratio to poly(.sup.imDNP-L-histidine)-folate (FIG. 3C) in
THF. The reaction was carried out for 2 days, and then the product
was collected by precipitation after adding excess n-hexane. The
product was then dialysed (Spectra/Por; MW cut off 6,000) to remove
unreacted ingredients. The product was then freeze-dried. To remove
the DNP group, the polymer was dissolved in dimethylformamide (DMF)
and then 2-mercaptoethanol was added (40-fold mole ratio excess) to
DMF with TEA (1 drop) (FIG. 3D). DMF/diethyl ether
recrystallization was twice repeated to remove
2-mercaptoethanol.
Cell Experiment with Sensitive MCF-7
[0074] Mixed polymeric micelles of
folate-poly(L-histidine)-PEG-PLLA and PolyHis-PEG were formed by
the dialysis method. The mixed polymeric micelles showed higher
cell cytotoxicity at tumor pH than at normal pH (FIGS. 4A-B). This
result indicates that interaction with and internalization of the
micelles into a selected breast tumor cell line of MCF-7 increased
as pH decreased.
Biotin/polyHis/PEG/PLLA
[0075] For the amination of biotin (FIG. 5A), biotin (1 mmol) was
activated with DCC (1.25 mmol) and NHS (1.5 mmol) in DMF.
Biotin-NHS was collected after recrystallization (DMF/diethyl
ether). Biotin-NHS (1 mmol) was dissolved in DMF and then mixed
with ethylene diamine (20 mmol) plus 500 .mu.g pyridine and allowed
to react at room temperature overnight. The reaction was confirmed
by thin layer chromatograph (TLC) analysis (silica gel plate,
2-propanol/chloroform=70/30 vol %). The product was dissolved in 1
N NaOH and then biotin-amine was precipitated and collected by
filtration. The product was also dissolved in 2 N HCl and then
filtered to remove insoluble materials. The solution was
freeze-dried for 3 days. For preparation of salt-detached
biotin-amine, biotin-amine was mixed with one drop of TEA and
recrystallized by adding excess diethyl ether. Incorporation into
block copolymers was carried out as described above and as
summarized in FIG. 5B.
pH-Dependent Aggregation of Carrier 4 Interaction with Avidin
[0076] The aggregation behavior of Carrier 4 by interaction with
avidin in solution was studied using turbidity change at various
pHs (FIG. 6). Avidin has four binding sites and a high binding
affinity for biotin. On decreasing the pH from 7.2 to 6.0,
turbidity changed slightly for Carrier 1, however, a significant
change was observed for Carrier 4. This change of turbidity was
attributed to the aggregation of nanoparticles by the interactions
between avidin and biotin, which was exposed at the nanoparticle's
surface due to deprotonization of the poly(His) attached to the
biotin moiety. When Carrier 4 was prepared at pH 8.0, the outer
shell was composed of PEG. Thus, such carrier showed high stability
above pH 7.4. However, below pH 7.0 the protonized poly(His)1K led
to exposing of the biotin ligand, resulting accessibility for
interaction with avidin.
Flow Cytometry Analysis of Carrier 4 at pH 7.2 and 7.0
[0077] For a more detailed observation of the interaction between
the nanoparticles and the cells, Carrier 4 was loaded with
adriamycin (ADR) as a fluorescence probe, and the interaction with
cells was monitored by flow cytometry analysis. At pH 7.0 Carrier 4
showed more internalization than at pH 7.2, because the biotin was
shielded at pH 7.2, but exposed on the surface of micelles at pH
7.0 (FIG. 7).
Fluorescence Microscopy of MCf-7/ADR.sup.R Cells
[0078] Fluorescence microscopy of MCF-7 cells was used to visualize
this interaction. For Carrier 4, the luminescence images clearly
distinguished the pH effect. At pH 7.0 and pH 6.8, the cells
strongly luminesced, while at pH 7.4 and pH 7.2 the cells showed
vague emission. In addition, the images suggested that Carrier 4 at
pH 7.0 and pH 6.8 was internalized into the cells (FIGS. 8A-L).
[0079] The internalization of drug carriers is one of the important
routes to enhancing the cytotoxic effect of the drug. The higher
intracellular content of a drug taken up via endocytosis of a
carrier may remarkably amplify the cytotoxic effect against the
target cells, providing that the carrier structure remained intact
with a high drug loading content until intracellular localization.
This is unlikely with pH-sensitive liposomes, as is has been
reported that such liposomes, without a specific ligand, failed to
deliver the contents to the cytoplasm of cells.
[0080] To confirm the accompaniment of ADR with Carrier 4 into the
cytoplasm at pH 7.0 and 6.8, the internalization of FITC-labeled
Carrier 4 loaded with ADR was monitored. FIGS. 8A-L represent the
confocal micrographs of the cells after incubation with the
nanoparticles. The green color image from FITC-labeled Carrier 4,
red image from ADR, and orange color from blended (overlapped)
green and red, were observed in the cytoplasm. In the case of
Carrier 5, the green color image from FITC-labeled Carrier 4, red
image from ADR, and orange color from blended (overlapped) green
and red, were found near the cell membranes and in the cytoplasm
(FIGS. 9A-L). This result is due to the lack of fusogenic effect of
the PLLA-PEG copolymer.
pH-Responsive Cytotoxicity of Carrier 4
[0081] The in vitro cytotoxicity to MCF-7 cells at different pHs
(7.4, 7.2, 7.0 and 6.8) were compared in FIG. 10. At pH 7.4 and
7.2, there was no noticeable difference in the cytotoxicity of
Carrier 4 at the ADR concentration range of 1 ng/mL to 10,000
ng/mL. However, at pH 7.0 and 6.8, ADR-loaded Carrier 4 showed
significantly enhanced cytotoxicity.
[0082] In summary, these results support that at pH 7.0 and 6.8,
Carrier 4 was associated with the cells and internalized together
with the entrapped drug in the cytoplasm, via endocytic mechanism.
The improved interaction and internalization of Carrier 4 at tumor
pH led to the improvement of cytotoxicity.
* * * * *