FIELD OF INVENTION
The present invention generally relates to novel hedgehog functionalized complexes
having nanoparticles bound to Hedgehog protein useful as a novel targeting agent for
the delivery of therapeutic or diagnostic siibstance at desired site.
5
BACKGROUND
The current standard of cancer therapy is inadequate (Takebe, Harris, Warren, & Ivy,
20 1 I). The very nature of the therapy e~nployed remains non-specitic, prolonging the
patient's sitrvival but failing to cure the cancer, which inevitably leads to their relapse
10 later. The inadequacy of current therapy regimens stems from their inability to target
the cancer stem cells (CSC) which are responsible for the regrowth of the tumor
following chemotherapy andlor radiotherapy (Harris, Speranza, & Dansky Ullmann,
2012). Additionally, the systemic concentration of chemotherapy agent required to
kill cancer cells is usually toxic to normal cell. The treatment and diagnosis of cancer
15 (or any disease) would be greatly enhanced / expedited by developing the ability to
deliver diagnostic probes or therapeutic agents into specific cells and cellular
compartments. Hence, there is an urgent need to evaluate and develop such
modalities that coilld specifically target the cancer cells and cancer stem cells without
affecting healthy organs and tissues, and may also help in concentrating the drug
20 preferentially in cancer cells.
As per an estimate, more than 95% of all new potential therapeutics being developed
have poor phannacokinetics and biopharmaceutical properties(Koo, Rubinstein, &
Onyuksel, 2005). Though milltiple strategies are being explored and are in different
stages of development. including targeting specific receptors expressed in particular
25 cancers for targeted therapy 1 diagnostics, their applicability, specificity and overall
tolerability had been a cause of concern(Koo et at., 2005)(Shi, Votruba, Farokhzad, &
Langer, 20 lo).
There is dearth of targeting moieties that can specifically guide the delivery of
therapeutic or diagnostic agent at desired site (Koo et al., 2005; Shi et al., 2010). This
problem is enormous in case of cancer due to unavailability of specitlc molecular
beacons that could be targeted.
Tlioi~gha ni~mbero f factors/molecules expressed in tumors are being evaluated for
targeting pote~ltiaal nd are i~ndevr arioits stages of development, there is still a lot that
5 needs to be done in terms of specificity and coming up with molecules that could be
used successfi~llyf or tlie 'thera~iostics'(Bolliassani,S afaiyan, & Rafati, 201 I ) .
Cancer stemlprogenitor cells express mucli higher level of receptors as opposed to
normal stem cells making the receptor based therapy more specific and target
oriented (preferential targeting of CSCs). This degree of specificity is not possible
10 with currently approved signaling inhibitors for treating cancer such as GDC0449 etc.
or by drugs i~nder various stages of development that target various steps in HH
signaling pathway(Takebe et al., 201 I) (e.g. GANTs).
Self-renewal signaling pathway Hedgehog (HH), WNT and NOTCH control the
proliferation, survival and 'sternness' of resident stem cells (SC) in many different
15 tissues (Beacliy. Karhadkar, & Berman, 2004)(Takebe et al., 201 I). These SC
maintain the liotneostasis through continilously replenishing the loss of cells in any
given tissue architecture. In cancers, these pathways are differentially attributed to
survival of CSC that is responsible for normal growth of cancer as well as relapse
after therapy, arid metastasis (Beachy et al., 2004)(Takebe et al., 201 1). HH signaling
20 is partici~larlym ore important as it has been found upregulated in >25% of all cancers
and suggested to be tlie key in survival and chemo-resistance of CSC that ultilnately
results in tumor relapse after therapy. The HH signaling is activated in cancer by
either binding of HH ligands [i.e. Sonic Hedgehog(SHH), Indian Hedgehog(lHH),
Dessert Hedgehog (DHH)] to its receptor Patched (PTCH; primarily PTCHI)
25 (Ingliam & Mcmalion, 200I)(Singh, Goetz, & Robbins, 2006) as in cancers of lung,
pancreas. liver prostate, or mutational activation of the pathway components
downstrea~n of HH ligand as in Basal cell carcinoma, rliabdomyosarcoma, and
medulloblastoma (Takebe et al., 201 1). Lung cancer is the leading cause of cancer
related deaths accounting for -20% of all cancer deaths. HH signaling is found
upregulated in upto 90% lung cancer cases (Yuan et al., 2007)(Singh et al., 201 1)
exclusively through HH ligand overexpression and its binding to receptor PTCH, and
it is shown to be responsible for the growth, tumorigenicity and 'sternness' by us and
others and more importantly it is upregulated in CSC (Bertolini et al., 2009). Since,
5 PTCHI is both receptor of HH ligand and main target gene of HH signaling, its
expression level gives a gauge of the pathway activity that can be harnessed to
selectively target this signaling and the cells expressing PTCH including that in CSC
for cancer 'theranostics'. Tl~us, PTCH seeking HH ligand can be effectively used to
target the delivery of specitic cargo including therapeutics (e.g. nanocon.jugates of
10 drugs, siRNA, and photosensitization agents) and diagnostictimaging agents (e.g.
contrast agents, fluorescent dyes) to HH activated cancer cell and CSC.
BRIEF DESCRIP'IION 01' THE DRAWINGS
Figure 1 shows the purification of HH ligand SHH from human SHH expressing
15 HEK cells. The SHH was purified using anti-SHH Ab and analyzed on 12% SDS
PAGE followed by im~nunoblotting.
Figure 2 presents a general outline of making Hedgehog (HH) ligand functionalized
nanoparticle for 'theranostics'.
Figure 3 illustrates the functionalization of gold nanoparticles with HH ligand causes
20 its targeted delivery to I'TCH (PTCH I) overexpressing cells. The 20nm gold particles
coupled with SIiI1-RSA were incubated with PTCH 1 expressing cells. SHH was used
as a representative HH ligand.
Figure 4 shows the targeting of SHH functionalized gold nanoparticles to be receptor
specitic. The attenuation of I'TCH I expression by PTCH I -siRNA decreased the
25 uptake of SHIH fi~nctionalized gold nanoparticles. The 20 nm gold particles coupled
with HH-BSA alone were incubated with 3T3 cells where endogenous PTCHl
expression was attenuated at different levels.
1 0 DEC 2013
DESCRIPTION 01' TIIE INVEN'I'ION
The present invention obviates the aforesaid problems of inherent lack of desired
specificity in various 'theranostic' agents and provides novel Hedgehog
functionalized cotnplexes useful as a novel targeting agent for the delivery of
5 therapeutic or diagnostic substance at desired site. In another aspect the present
invention discloses the Hedgehog (HH) ligand as a novel targeting agent for various
conditions including that of cancer or cancer stem cells where HH receptor PTCH I is
differentially expressed.
The present invention also relates to Hedgehog (HH) ligand based targeted therapy
10 for HH receptor PTCH expressing cancer cells or CSC. Such targeted therapy may
eventually eliminate the side effects associated with the current cancer therapy and
cure the cancer by eliminating CSC and therefore tremendously improve the outcome
of cancer therapeutics and diagnostics. Additionally, such HH ligand based delivery
vehicle could be elnployed for delivery of therapeutic or diagnostic cargo to other
15 conditions where HH signaling receptor P'TCH is involved or differentially expressed
1e.g. wound repair, treatment of retinal damage, neuronal injury or degeneration (e.g.
Parkinson's disease) etc.]
The present invention relates to Hedgehog ligands (e.g. SHH) which are
overexpressed concomitantly with its receptor PTCH l in a number of pathological
20 conditions i~icluding cancer can be effectively used to target the delivery of
diagnostic and therapeutic agents to specific cells as demonstrated by PTCH I dose
dependent delivery of SHH functionalized gold nanoparticles. Similar methodologies
could be employed for preparing hedgehog functionalized nanoparticles wherein the
~ianopa~-ticleasre dendritners, poly lactide-co glycolic acid (PLGA), polycefins or
25 Lipid-polyethyle~ii~ni~ihey brid nanocarriers, made of metals, nonmetals or
composites thereof etc. The nanoparticles may also be optionally loaded with a
tluorescent dye andlor a cytotoxic agent.
In a preferred embodiment, the present invention relates to a novel hedgehog
functionalized complex comprising: a nanoparticle; a hedgehog ligand; and a linker
molecule having a first end bound to tlie nanopasticle and a second end bound to the
hedgehog l igand.
In anotlier embodiment tlie present invention relates to the novel hedgehog
fi~nctionalized complex. wherein the nanoparticle is a metal nanoparticle, lion-metal
5 nanoparticle, composite nanopasticle, polymer nanoparticle or mixtures thereof.
In another embodiment the present invention relates to the novel hedgehog
functionalized complex, wherein the metal nanoparticle comprises at least one of
aluminum. iron. silver, zinc, gold, copper, cobalt, nickel, platinum, manganese,
rhodium, ruthenium. palladium, titanium, vanadium, chromium, molybdenum,
10 cadmium, mercury, calcium, zirconium, iridium, and oxides thereof, preferably gold.
In anotlier embodiment the present invention relates to the novel hedgehog
functionalized complex, wherein the nanoparticle has an average particle size ranging
from I to I00 nm, preferably I - 50 nm, more preferably 10-40 nm, more preferably
20 nm.
15 111 another embodiment tlie present invention relates to the novel hedgehog
functionalized complex, wherein the functionalized nanoparticle comprises
nanoparticles of polymers such as dendrimers, poly lactide-co glycolic acid (PLGA).
polycetins or Lipid-polyetliylenimine hybrid nanocarriers, or other suitable polymers
or materials.
20 111 another embodiment the present invention relates to the novel hedgehog
functionalized coinplex, wherein the linker rnolecule is serum albumin, selected from
bovine serum albumin and Iiurnan serum albumin.
In another embodiment tlie present inventio~i relates to the novel hedgehog
functionalized complex, wherein the linker molecule is bovine serum albumin and the
25 nanoparticle is gold nanoparticle.
In another embodiment tlie present invention relates to the novel hedgehog
functionalized complex, wherein tlie hedgehog (HH) ligand is Sonic Hedgehog
(SHH), Indian Hedgehog (IHH), or Dessert Hedgehog (DHH).
In another embodiment the present invention relates to the novel hedgehog
functionalized complex, wherein the hedgehog functionalized complex is loaded with
a fl~lorescentd ye and/or a cytotoxic agent.
Yet another preferred embodiment of the present invention relates to a method for the
5 preparation of the hedgehog functionalized complex, said method comprising:
preparing linker-hedgehog conjugates by mixing purified hedgehog ligand with a
linker in presence of a cross linking agent; and mixing the linker-hedgehog
conjugates with nanoparticles to form a hedgehog functionalized complex.
In another embodiment the present invention relates to the method for tlie preparation
10 of the hedgehog filnctionalized complex, wherein the cross linking agent is a
bifi~nctio~icarlo ss linking agent or a multifunctional cross linking agent.
In another embodiment the present invention relates to the method of preparation of
hedgehog functionalized complex, wherein the bifunctional cross linking agent is
selected from the group consisting of Disuccinimidyl suberate (DSS), 3-
15 maleimidobenzoic acid N-hydroxysuccinimideester (MBS) or any variations thereof.
In another embodiment tlie present invention relates to the method for the preparation
of the liedgeliog filnctionalized complex, wherein the puritied hedgehog ligand and
the linker are mixed together in a predetermined ratio such that the resulting linkerhedgehog
cotijugates prepared have a conjugation ratio in the range of between 1 : 1
20 and 1.4, preferably, l : I .
In another embodiment the present invention relates to the method for the preparation
of tlie Iledgeliog functionalized complex, wherein the nanoparticle is selected from
the group consisting of a metal nanoparticle, non-metal nanoparticle, composite
nanoparticle, polymer nanopal-ticle or mixtures thereof.
25 In another embodiment tlie present invention relates to the method for the preparation
of the hedgehog fi~nctionalized complex, wherein the metal nanoparticle comprises at
least one of aluminum, iron, silver, zinc, gold, copper, cobalt, nickel, platinum,
manganese. rliodii~m. ri~theniutn, palladium, titanium, vanadium, chromium,
molybdenum. cadniium, mercury, calcium, zirconium, iridium, and oxides thereof.
In another embodiment the present invention relates to the method for the preparation
of the hedgehog fi~nctionalized complex, wherein the functionalized nanoparticle
comprises a polymer such as dendrimers, poly lactide-co glycolic acid (PLGA),
polyceflns or Lipid-polyethylenimine hybrid nanocarriers, or other suitable polymers
5 or materials.
In another embodilnent the present invention relates to the method for the preparation
of the hedgehog functionalized complex, wherein the hedgehog ligand is Sonic
Hedgehog (SHH). Indian Hedgehog (IHH), or Dessert Hedgehog (DHH).
In another embodiment tlie present invention relates to the method for the preparation
10 of the hedgehog fi~nctionalized complex, wherein the linker molecule is a serum
albumin, preferably selected from bovine serum albumin and human serum albumin.
In anotlier embodiment tlie present invention relates to the method for the preparation
of the hedgehog fi~nctionalized complex, wherein the hedgehog ligand is Sonic
Hedgehog, tlie linker is bovine/human serum albumin and nanoparticle is that of gold.
15 In another embodiment the present invention relates to the method for the preparation
of the hedgehog functionalized complex, wherein the method further comprises
loading of the hedgehog filnctionalized complex with a fluorescent dye and/or a
cytotoxic agent.
EXAMPLE
20 Materials: Sterile Ultrapure deionized water ( 1 8-MR/cm2) was used for making all
buffers. Citrate capped Gold nanoparticles (20 nm diameter), Bovine serum albumin
(BSA) (M W: 67000). Dis~~ccinitnidsyul berate (DSS), 3-Maleimidobenzonicacid NIiydroxysuccini~
iiide ester (MBS) and dithiothreitol (DTT) of highest purity were
obtained from colnrnercial suppliers. Sephadex (310, G25, and G50 (for size
25 exclusion chromatography) were purchased from Sigma. Centricons (YM-3, YM-30)
and M icrocon Y M- 100 were purchased from Millipore. 7.5 and 12% SDS-PAGE gels
were made in-house. Protein concentration was estimated using protein assay kit
from Bio-Rad.
Hedgehog ligun~l:T he Iiuman SHH expression construct (pcDNA-SHH) or a control
(ctrl) was trarisfected into HEK293 cells using Lipofectamine 2000 as per
manufacturer's recommendation. Two days later medium was changed to serum free
growth medium. The following day growth medium was collected, cell debris
removed by centrifuging at 9000g for 60 min, concentrated using YM-3 and purified
using anti-SHH aftinity chromatography essentially as described in the prior art
(Taipale et al., 2000) followed by separation on 12% Sodium Dodecyl sulfate
Polyacrylamide Gel Electrophoresis (SDS-PAGE) and immunoblotting with anti-
SHH Ab (H- 160, Santa Cruz Biotechnology).
Preparation of lledgehog functionalized Gold Nanoparticle (MH-BSA-Au-NP):
Hedgehog-gold particle complexes were assembled by con-jugating Hedgehog to
bovine serum albumin (BSA) and then attaching BSA-peptide conjugates to gold
nanoparticles (Figure I ). To prepare BSA-Hedgehog conjugates, BSA was mixed
with SHH, and a bifunctional cross linking agent [e.g. Disuccinimidyl suberate
(DSS), 3-maleimidobenzoic acid N-hydroxysuccinimideester (MBS)] in all
empirically determined molar ratio that mostly conjugated BSA and SHH in 1 : 1-4
ratio. After purification by gel filtration on Sephadex C50, the efficiency of
conjugation was estimated by mobility shift assay on 7.5% SDS-PAGE. The SHH
con-jugated BSA (SHH-BSA) was added to 20 nm gold nanoparticles to generate HHBSA-
ALI nanopat-ticle complexes (i.e. SHH- BSA-AuNP).
Characterization of HH-BSA-AuNP: The nanoparticle complexes were
characterized for bo~~nHdH -BSA, dispersity, structure, size, zetapotential sing
established techniques (i.e. Protein assay, SEM, Zetasizer ZS90) essentially as
described (Tkaclienko et al., 2004)(TkacIienko et al., 2003).
Cell Culture: Mouse 3T3/NIH, and human (e.g. HOP62, A.549, H23) cell lines were
obtained from American Type Culture Collection (Rockville, MD) and maintained in
Dulbecco's Modified Eagles Medium (DMEM) or RPMI-1640 growth media without
addition of antibiotics at 37 "C in controlled CO. atmosphere.
Modulation of Hedgehog receptor expression in mammalian cells /specificity of
HH-BSA-AuNP uptake: The cell lines were plated at different cell densities (0.2-
0.3xl0"cells) ill 6 well plates. In case of Hedgehog receptor PTCH I overexpression,
the following day cells were transfected with different amount of PTCH 1 expression
constritct or an insertless control pcDNA3.1 vector using Lipofectamine 2000 as per
manufacturer's directions. For Hedgehog receptor PTCH 1 expression attenuation the
cells were transfected with two validated PTCH 1 siRNA (I-5pmol) or a FlTC labeled
scrambled Control siRNA (Ctrl siRNA) as per manufacturer's direction. Two days
later the transfection efficiency was determined in a parallel experiment based on
GFP or FITC presence inside the cells. The modulation in PTCHl expression was
detected by imniunoblotting of the cell I ysate (anti-PTCH Ab; C-20, Santa Cruz
Biotechnology), and quantitated using Image J software.
For investigating the cellular uptake, the 3T3 cell expressing HH receptor PTCHI
(overexpressedlattenuated) were plated on glass coverslips and grown to 75%
confluency in 12-well plates and then incubated with nanoparticle conjugate HHBSA-
AuNP (50-150 pM) for various times (e.g. 4 hrs) in 10% Fetal Bovine Serum
(FBS) containing medium. Similarly, the targeting potential of HH in delivery of
nanoparticles/nanocarriers (HH- BSA-AuNP) to common lung cancer cell lines (e.g.
HOP62, A549, H23) expressing different level of HH receptor PTCHl were also
evaluated. At the end of the experiment the coverslips were rinsed extensively with
phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 15 min
at room temperature and then rehydrated in PBS. Following fixation, the cover slips
with cells were mounted onto glass slides with mounting media and allowed to dry
overnight prior to microscopy analysis. Similarly, the targeting potential of HH in
delivery of na~ioparticles/~ia~iocarriteor sc ommon lung cancer cell lines (e.g. HOP62,
A549, H23) expressing different level of HH receptor PTCHI was also evaluated.
Microscopy: The delivery of nanoparticle was assessed using differential
5 interference contrast microscopy (DIC microscopy) as described in the prior art
(Tkachenko et al., 2004). A software-based manual control of color balance and
contrast enhancement on live and captured images was performed wherever needed
for better visitalization. Using image processing software (e.g. ImageJ) relative fold
increase in the delivery of HH functionalized nanoparticles (e.g. HH-BSA-AuNP)
10 was assessed. TI-ansmission Electron Microscopy (TEM) analysis of the samples was
performed as per established protocols (Tkachenko et al., 2004).
Evaluation of targeted delivery potential of Hedgehog functionalized
nanoparticlelcarrier in vitro: The mouse cell line 3T3 expressing different levels of
15 HH receptor PTCH I (expression modulated by PTCHI overexpression as well as
knockdown using siRNA1shRNA to PTCH I) was evaluated for the specificity and
targeted delivery potential of HH fi~nctionalizedn anoparticles (i.e. HH- BSA-AuNP).
The delivery of nanoparticles was assessed using differential interference contrast
microscopy as well as TEM (Tkachenko et al., 2004). The targeted delivery,
20 therapeutic potential and suitability for photodynamic therapy were assessed sing
cell proliferation and apoptosis assays essentially as described in the prior art (Singh
et al., 201 ])(Tan et al., 2012)(Luo, Zliang, Su, Cheng, & Shi, 201 I). Similarly, the
targeting potential of HH in delivery of nanoparticles/nanocarriers to common lung
cancer cell lines (e.g. HOP62, A549, H23) expressing different level of HH receptor
25 PTCH was also evaluated.
Results:
Purification of IrHH: The recombinant human SHH purified from human SHH
expressing HEK cells essentially as described in the prior art (Taipale et al., 2000)was
further concentrated using Centricons. The tinal protein preparation was analyzed by
5 12% Sodium Dodecyl sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and
immunoblotted for content and purity (Figure I). The protein preparation was found
to be > 95% pure. The concentration of protein was determined by BCA assay using
BSA as reference. This purified concentrated SHH preparation was further used for
cotijugation to BSA then to gold nanoparticles (AuNP).
10 Preparation of SHH decorated Gold Nanoparticle (SHH-BSA-AuNP): Human
SHH - gold nanoparticle complexes were assembled by first con.jugating SHH to
bovine serum albumin (BSA) followed by attaching BSA-SHH conjugates to gold
nanoparticles (Figure 2). To prepare BSA-Hedgehog conjugates, BSA was mixed
with SHH, and a bifilnctional cross linking agent [e.g. Disuccinimidyl suberate
15 (DSS), 3-maleimidobenzoic acid N-hydroxysuccinimideester (MBS)] in an
empirically determined molar ratio that mostly conjugated BSA and SHH in I:\-4
ratio. The HH conjugated BSA (HH-BSA) was added to 20nm gold nanoparticles to
generate HH-BSA-Au nanoparticle complexes (HH- BSA-AuNP). An empirically
determined ratio of SHH, BSA and bifunctional conjugating agent DSS resulted in
20 con~jugation of SHH to BSA in 1-4:l ratio. After purification by gel filtration on
Sephadex G50, the efficiency of conjugation was estimated by mobility shift assay on
SDS-PACE. The BSA-SHH corijugates were then attached to 20 nm diameter gold
particles in carbonate buffer (20 mM, pH 10.5) to generate HH-BSA-Au nanoparticle
colnplexes (HH- BSA-AuNP). Dynamic light scattering and transmission electron
25 microscopy revealed that BSA-HH conjugates add -9 nm to the hydrodynamic
diameter ofa 22 nm gold particle. Cellular uptake of HH-BSA-AuNP complexes was
primarily characterized in 3T3/NIH, and lung cancer cells (e.g. A549 HOP62 cells).
These cell lines were chosen based on the presence of functional HH signaling and
differential PTCH 1 expression.
Nanoparticle delivety to Cells: The delivery of nanoparticle to 3T3 cells (and others)
was found to be dependent on PTCH l expression (see Table 1 and 2). The ectopic
5 expression of PTCH 1 increased the uptake of HH-BSA-AuNP (compare Ctrl plasmid
transfected cells with the cells transfected with different amounts of
PTCH l expression plasmid i.e. PTCH I. 1-3 in Table 1. This increase in nanoparticle
uptake was dose dependent and a 20-30 fold increase in PTCHl expression resulted
in upto 100 fold increase in nanoparticle uptake. Similarly, attenuation of endogenous
10 PTCHl expression in 3T3 resulted in diminished HH-BSA-AuNP uptake (Table 2).
This conclusively demonstrates the feasibility and utility of HH ligand in targeting
HH-receptor PTCH l expressing cells. Similar PTCH l dependence of HH-BSAAuNP
uptake was observed in lung cancer cell lines (e.g. HOP62, A549) which are
known to express different levels of PTCH 1.
15 The delivered nanoparticles mostly localized in cytoplasm and appeared to be
clustered in endosome as per TEM analysis (data not shown). This was expected as
PTCH I is known to get endocytosed on HH ligand binding.
Although the foregoing invention has been described in terms of certain preferred
embodiliients, other embodiments will be apparent to those of ordinary skill in the art
20 from the disclositre herein. Additionally other combinations, omissions, substitutions,
and modifications will be apparent to the skilled artisan in view of the disclosure
herein. For example, although the present invention has been described using the
example of Hedgehog functionalized Gold Nanoparticle (HH-BSA-Au-NP), various
other kinds of nanoparticles linkers may also be utilized for preparing the Hedgehog
25 functionalized colnplexes of the present invention. All such variations of the
hedgehog functionalized complexes are considered to be within the scope of the
present invention. It is contemplated that various aspects and features of the invention
described can be practiced separately, combined together, or substituted for one
another, and a variety of combination and sub-combinations of the features and
aspects can be made and still fall within the scope of the invention. Accordingly, the
5 present invention is not intended to be limited by the recitation of the preferred
embodimelits.
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I Claim,
A novel hedgehog functionalized complex comprising: a nanoparticle; a
hedgehog ligand; and a linker molecule having a first end bound to the
nanoparticle and a second end bound to the hedgehog ligand.
TIie novel hedgehog fulictionalized complex as claimed in claim 1, wherein
the nanoparticle is a metal nanoparticle, non-metal nanoparticle, composite
nanoparticle, polymer nanopasticles or mixtures thereof.
The novel hedgehog functionalized complex as claimed in claim 2, wherein
the metal nanoparticle comprises at least one of aluminum, iron, silver, zinc,
gold, copper, cobalt, nickel, platinum, manganese, rhodium, ruthenium,
palladium, titanium, vanadium, chromium, molybdenum, cadmium, mercury,
calcium, zirconium, iridium, and oxides thereof, preferably gold.
The novel hedgehog functionalized complex as claimed in claim 1, wherein
the rianoparticle comprises nanopasticles of dendrimers, poly lactide-co
glycolic acid (PLGA), polycetins or Lipid-polyethylenimine hybrid
nanocarriers.
The novel Iiedgeliog functionalized complex as claimed in claim 1, wherein
the linker molecule is serum albumin, selected from bovine serum albumin
and human serum albumin.
The novel hedgehog functio~ializedc omplex as claimed in claim 1, wherein
the linker rnolecule is bovine serum albumin and the nanoparticle is gold
nanoparticle.
A method for the preparation of the hedgehog functionalized complex as
claimed in claim 1, said method comprising: preparing linker-hedgehog
con-jugates by mixing purified hedgehog ligand with a linker in presence of a
cross linking agent, wherein the cross linking agent is a hi-functional cross
linking agent or a multifunctio~~carlo ss linking agent; and mixing the linkerhedgehog
col?jugates with nanoparticles to form a hedgehog functionalized
complex.
8. The method as claimed in claim 7, wherein the bifunctional cross linking
agent is selected from the group consisting of Disuccinimidyl suberate (DSS),
5 3-maleimidobenzoic acid N-hydroxysuccinimideester (MBS) or combinations
thereof.
9. The method as claimed in claim 7, wherein the purified hedgehog ligand and
the linker are mixed together in a predetermined ratio such that the resulting
linker-hedgehog con.jugates prepared have a con-jugation ratio in the range of
10 between 1 : I and 1 :4, preferably, 1 : I .
10. The method for the preparation of the hedgehog functionalized complex as
claimed in claim 1, wherein the hedgehog ligand is Sonic Hedgehog, the
linker is bovine serum albumin and nanoparticles is gold nanoparticles.