Field of Invention:
This invention relates to drug delivery formulation of rapamycin in the form of
polymeric nanoparticles for cancer therapy.
Background of the Invention:
Cancer is one of the most devastating micro evolutionary processes leading to its
high rate of mortality. The disease clawed the entire body systems leaving little
to save the patient. Rapamycin, a macrolide fungicide first isolated from
Styptomyces hygroscopicus\x\ the early 1970s, used in renal transplantation in
1990 as an immunosuppressant, has recently been shown impressive anti-tumor
activity by blocking the translation of proteins required for cell cycle progression
from G1 to S phase and its potentiality arrests the cell growth derived form
pancreatic cancer, breast cancer, prostrate cancer, lung cancer,
rehabdomyosarcoma, glioblastoma, neuroblastoma, osteosarcoma, leukemia and
β-cell lymphoma. Rapamycin shows its antitumor activity by binding
intracellularly to the immunophilin FK506-binding protein (FKBP12) and the
resultant complex then inhibits the mammalian target of rapamycin (m-TOR) or
FRAP, a protein kinase belongs to phosphoinositide 3- kinase (P13K) super
family, m TOR, a serine/threonine protein kinase regulates the eukaryotic cell
growth, protein synthesis and proliferation . m TOR inhibition blocks the

signals which induce the phosphorylation of the 70 KDa, 40S ribosomal protein
kinase (p70S6K) and the eukaryotic initiation factor 4-E binding protein -1 (4E-BP
1) leading to GI arrest in most cell types and to p53-independent apoptosis in
. others [6,7]. Though rapamycin has excellent anticancer effects but the clinical
application has been limited due to very poor solubility in water (2.6ug/ml), low
oral bioavailability i.e. not absorbable in gastrointestinal tract, a number of
undesirable side effect like low accessibility to tumor tissues, nonspecific delivery
, multidrug resistance due to the metabolism of enzymes of P450 3A subfamily
and over expression of plasma membrane transporter p-glycoprotein (p-gp)
present at apical membrane which effluxes the drug into intestinal lumen
restricting their absorption into blood, increased drug toxicity and adverse side
effects to the normal tissue .
In clinical pharmacology one of the major problems associated with poor water
soluble (hydrophobic) active drugs in systematic delivery. In order to increase
the potency of these promising drugs new methodologies have been developed
to overcome the lacunae of conventional drug treatment. For solubilising the
hydrophobic drugs, the conventional excipients like Cremophore EL, Tween
(Polysorbate)-80 has been used. But these solvents caused-incidental adverse
effects like severe hypersensitivity, neurotoxicity and nephro toxicity etc. Till
date, NPs based therapeutics is one such approach designed to enhance the
efficacy of the drug which results sustained drug release within the tissue over
an extended period reducing the lethal side effects caused by the drug. Most of
the formulations developed, are administered intra venous and not suitable for
oral delivery. Oral chemotherapy, a challenge in oncology can be

attractive to patients as cancer patient appreciate easy, convenient and non-
invasive home- based administration of drugs in form of pill. These pills by an
oral route may get better quality of patients' life in both early and later stage of
cancer, those unable to accept any. treatment due to weakness. Some
randomized trials showed oral delivery reduces the drug related undesirable
effects (like diarrhea, nausea/vomiting etc) without compromising on efficacy. It
provides appropriate concentration of drug in circulation resulting sustained
release of drug to cancer cells and produces a much better efficacy than current
clinical administration like injection and infusion. Recently, cubic phase
nanoparticulate systems have received much more attention for sustain release
of both hydrophilic and hydrophobic drugs in drug delivery research. This phase
has the ability to solubilise the hydrophilic, hydrophobic and amphiphilic
molecules. In this view, GMO is a synthetic lipid amphiphilic molecule approved
by food and drug administration was used for controlled drug delivery in the
form of cubic phase nanoparticles. The cqbic phase formed in water is a three
dimensional network of curved lipid bilayers separated by intricate network of
water channels. Many types of polymers has been used for NPs formation, but
the requirements of the biocompatibility and biodegradability have limited the
choice of polymers used in clinical applications. Some representatives of such
material are non ionic block copolymer Pluronic F-127 and vitamin -E TPGS.
These have gained much interest for providing stability and chemical
functionalization to NPs in drug delivery system. Previous studies support the fact
that Pluronic F-127 can enhance the drug transport by effective passive targeting
towards cancerous tissues, as well as sensitize the multi drug resistance tumor to
various neoplastic agent and inhibit the drug efflux transporter. Similarly, vitamin
-E, TPGS is a well known

biocompatible natural polymer used as solubliser, emulsifier and most often
served a vehicle for lipid-based drug delivery formulations due to its surface
active properties (hydrophilic polar head and lipophilic alkyl tail). Furthermore, it
acts as an absorption enhancer and bioavailability promoter which could facilitate
the drug loaded NPs to transport the drug across the gastrointestinal barrier.
Besides serving oral enhancer, in recent years it is reported to be one of the
most effective p-gp inhibitor amongst the surfactants.
In this study, we describe a simple method of preparation and characterization of
rapamycin loaded GMO NPs blended with" amphiphilic block copolymer pluronic F-
127 and a natural emulsifier vitamin E-TPGS, would enhance oral bioavilibility of
the hydrophobic drug rapamycin. Cytotoixity, cellular uptake , sub-cellular
localization and blocking of AKT/mTOR pathway were studied in MIA PaCa cells
and confirmed that rapamycin loaded GMO NPs were more effective in inhibiting
the tumor cell proliferation due to their higher uptake as demonstrated by flow
cytometry and confocal microscopy. Further, we have reported the
pharmacokinetics profile on an orally administered rapamycin encapsulated in
GMO NPs and its biocompatibility was tested in animal model using Balb/c mice.
OBJECTS OF THE INVENTION:
An object of this invention is to propose a drug delivery formulation of rapamycin
in the form of polymeric nanoparticles.
Another object of this invention is to propose a dry drug delivery formulation of
rapamycin when the average zeta potential of the nano particles is 31.51± 3.5
with good aqueous dispersibility.

Further object of this invention is to propose a process of preparation of a water
soluble rapamycin loaded nanoparticles with high encapsulation efficiency of
92%.
Still another object of this invention is to propose a drug delivery formulation of
rapamycin which allows administration of high dose of rapamycin sustainable
over an extended period.
Still further object of this invention is to propose a drug delivery formulation of
rapamycin which has a sustained release of rapamycin entrapped inside the nano
particles suitable for oral drug delivery.
Yet another object of this invention is to propose a drug delivery formulation of
. rapamycin with enhanced bioavailability.
Brief Description of the Invention:
According to this invention, there is provided an anticancer rapamycin drug
delivery formulation in the form of polymeric nano particles for cancer therapy,
having absorption enhancer & superior oral drug bioavailability properties.
In accordance with this invention there is provided a method for preparing a dry
delivery formulation of rapamycin in the form of polymeric nano particles
comprising the steps of:
Subjecting the said GMO mixture to the step of emulsification with pluronic
solution,
Emulsifying the resultant solution once again with solution of vitamin - E TPGS;
Lyophilizing the final emulsion by freeze drying to produce lyophilized powder
having rapamycin

Brief description of the accompanying Drawings:
Figure 1
(a) Mean particle size of rapamycin loaded GMO NPs by using a particle
size analyzer. (Hydrodynamic diameter = 88 ± 6nm, n=3)
(b) Size distribution of rapamycin loaded GMO NPs by Atomic force
microscopy (AFM).
(c) The representative picture of rapamycin, loaded GMO NPs as
obtained from transmission electron microscopy . (bar= 0.2um)
Figure 2
(a) FT-IR spectra of (i) native rapamycin, (ii) rapamycin loaded GMO
NPs and (iii) void GMO NPs.
(b) DSC thermogram of (i) native rapamycin, rapamycin loaded (ii) GMO
NPs and (iii) void GMO NPs.
(c) X- Ray Diffraction study of (i) void GMO NPs (ii) rapamycin loaded
GMO NPs (iii) native rapamycin.
(d) In vitro release kinetics of rapamycin from GMO NPs in PBS (0.01 M,
pH 7.4) at 37 °C.
Figure 3
(a) Quantitative cellular uptake study of native 6-coumarin (Green) and
6- Coumarjn loaded GMO NPs (Pink) on MIA PaCa cell line by flow
cytometry.
(b) Histogram analysis of quantitative cellular uptake study of native 6-
coumarin and 6-coumarin loaded GMO NPs. Data as mean ±s.e.m,
=3(**) p<0.005.

Figure 4
(a) At various time intervals the sub-cellular localization of 6-coumarin
loaded GMO NPs were studied in MIA PaCa cell line using orange red
propidium iodide (PI) (for nucleus staining) by confocal
microscopy.
(b) Nuclear co-localization of 6-coumarin loaded GMO NPs was
observed by combining green florescent dye of 6-coumarin with
orange-red PI following 1 hr exposure in MIA PaCa cell line.
Figure 5
Dose dependent cytotaxicity study of native rapamycin (•), rapamycin
loaded GMO NPs (▲) and void, GMO NPs (■) in different cell lines.
cytotoxicity was measured at 5 days by MTT assay. Percentage of
inhibition was done respect to controls. Data as mean ± s.e.m., = 6.
(**) p<0.005 and (*) p< 0.05.
Figure 6
(a) Loss of mitochondrial membrane potential (MMP) studied •
in MIA PaCa cells due to the effect of native rapamycin
and rapamycin loaded GMO NPs (100 ng/ml) by flow
cytometry after 48 hr of treatment. Top right: live cells;
bottom right: cell with loss of membrane potential.
(b) Induction of apoptosis in MIA PaCa cell line by native
rapamycin and rapamycin loaded GMO NPs (50ng/ml)

by flow cytometry after 48 hr of treatment, Top left :
necrotic cells ; top right: late apoptotic cells ; bottom left
;live cells ; bottom right: early apoptotic cells.
Figure 7
Inhibition of p-Akt and downstream targets of mTOR expressions
Induce apoptosis in MIA PaCa cells as confirmed by
immunoblotting analysis.
Figure 8
(a) Toxicity profile of void GMO NPs administered at a dose of
500mg/kg to BALB/c mice and kept the animals for
observation up to 30 days, (i) Oral administration of void
GMO NP revealed no weight loss in BALB /mice, (ii) After 30
days of administration, animals were sacrificed and
histological profiles were taken in to study. Representative
photomicrographs of lung, kidney, intestine and liver by H &
E staining.
(b) In vivo pharmacokinetic studies of native rapamycin (■) and
Rapamycin loaded GMO NPs (•) (15mg/kg) in Balb /c mice
after oral administration. The inserted bar diagram showed
bioayailability of native rapamycin and rapamycin loaded
GMO NPs after 24 hr of drug administration. Data as mean
±s.e.m, =3. (**) p<0.005.

Detailed Description of the invention:
Preparation of Rapamycin loaded NPs
Rapamycin loaded GMO NPs were prepared by little modification of our pervious
protocol [20]. Briefly, 200mg of rapamycin was incorporated into the fluid phase
of GMO (1.75ml at 40° C) The above mixture was emulsified with 10 ml of
pluronic F-127 solution (10% w/v) by sonication, using a microtip probe
sonicator(Model : VCX750, Sonics and materials Inc, USA) set an amplitude 30%
for 2 min over an ice bath. The resultant solution was further emulsified with 10
ml of Vitamin -E TPGS (5% w/v) and sonicated for 2 min at amplitude 30 %
over an ice bath. The resultant nano particulate emulsion were frozen (-80 C°)
and lyophilized by freeze drying methods for six days (-80°C and < 10mm
mercury pressure, LYPHLOCKL, Labconco, Kanas City, MO) to get the lyophilized
powder for further use. To determine the cellular uptake NPs, the 6-coumarin
loaded GMO NPs were prepared using the above procedure except that 100 ug
of the dye was added to GMO prior to emulsion instead of rapamycin.
Physio-chemical characterization of rapamycin loaded NPs.
Particle size and zeta potential (ﯼ) measurements
The particle size and size distribution measurements were done by using a
Malvern Zetasizer Nano ZS (Malvern Instrument, UK) based on dynamic light
scattering. Briefly, ~ lmg/ml rapamycin loaded GMO Nps solution were prepared
in double distilled water and sonicated for 30 sec in an ice bath (VC 505,

Vibracell Sonics, Newtown USA). Size measurements were performed by
following the dilution of NPs suspension in Milli Q water at 25 ° C (lOOul diluted
to 1 ml). The same suspension was used for measuring the zeta potential at NPs.
All measurements were preformed in triplicates.
Atomic Force Microscopy (AFM)
The shape and surface morphology of rapamycin loaded GMO NPs were
investigated by atomic force microscopy (Nano III A, Vecco, USA). One drop of
lmg/ml rapamycin loaded GMO NPs solution was placed on freshly cleaved mica
and incubated for 5 min. To remove the unbound the NPs the surface was rinsed
with deionised water. The sample was air dried and mounted on the microscope
scanner. The shape was observed and imaged in noncontact mode with
frequency 312 KHz and scan speed 2 Hz.
Transmission Electron Microscopic Studies
The internal morphology was examined by Transmission Electron Microscopy
(TEM). In brief, - 1mg of GMO NPs was suspended in distilled water and
sonicated for 30 sec. A drop of this suspension was placed in carbon coated
copper TEM grid (150 mesh, Ted Pella Inc, Rodding, CA), negatively stained with
1% uranyl acetate (w/v) for 10min and allowed to air-dry. The samples were
imaged using a Philips 201 Transmission Electron Microscopy (Philips/FE Inc,
Manor, and NY) and visualized at 120 KV under microscope. The TEM
photograph was taken using the NIH imaged software.

Fourier Transform Infrared Spectroscopy (FT-IR)
This experiment was performed to investigate the chemical interactions between
rapamycin and the polymer matrix. Different samples (Void GMO NPs, native
rapamycin and rapamycin loaded NPs separately) were mixed with KBr (in ratio
1:10) and ground into fine powder using as agate mortar. The mixture was
pressed into a pellet with a pressure of 150 kg /cm2. The KBr pellet was
subjected for analysis using FT-IR Spectrometer (Perkin Elmer, Model spectrum
RX 1, USA) by averaging 32 interferograms with resolution 2 cm"1 in the range of
800 to 4000 cm"1-
Differential scanning Calirometry (DSC)
The physical status of rapamycin encapsulated in NPs was characted by using
DSC thermo gram analysis (STA 6000, Simultaneous Thermal Analyser, Parkin
Elmer, MA, and USA). Each sample-7 mg (native repemycin, void GMO NPs and
rapamycin loaded GMO NPs) were placed in a standard aluminum pan separately
and the samples were purged in DSC with pure dry nitrogen gas set at. a few
rate of 10ml/min, temperature speed 10° C /min. and the heat flow was recorded
from 30 to 3000 C.
X-ray Diffraction (XRD) Study
XRD analysis was carried out to know the crystallinity of the NP formulation. The
spectra were obtained by using X- ray diffractometer (D8 ADVANCE, Bruker AXS,
USA). The measurements were done at a voltage of 40 KV and 25 mA. The
scanned angle was set form 3°< 20 < 40°and the scan rate was 0.90min 1-

Quantifying entrapment efficiency of rapamycin by high performance
liquid chroma tog rapy (HPLC) method
The entrapment efficiency of rapamycin was determined as per previous
published protocol [26]. Accordingly, rapamycin loaded GMO NPs were dissolved
in acetonitrile (-1 mg/ml). The sample was then sonicated for 2 min at amplitude
30% in an ice bath (Model: VCX 750, Sonics and materials Inc, USA) and
centrifuged at 13, 800 rpm for 10 min at 4 C (SIGMA l-15k, Germany). The
obtained supernatant was analyzed using reverse phase icocratic mode (RP-
HPCL) system of Waters ™ 600, Waters Co. (Milford, MA, USA). For
quantification of drug, 20 ul supernatant was injected manually in the injection
port and analyzed using a mobile phase of acetionitrile: water in the ratio 80: 20
(v/v). The flow rate was 1 ml/min with a quaternary pump (M600E WATERS ™)
at 20° C with C 18 column (Nova - Pac, 159X 4.6 mm, internal diameter).
Rapamycin level was quantified by UV detection at 278 mm with dual wavelength
detector (2489). The amount of rapamycin in NPs was obtained form the peak
area correlated with the standard curve. All analysis was performed in triplicates.
The entrapment efficiency was calculated from the following equation:
Entrapment efficiency (%) = (Amount of rapamycin in NPs/ Amount of
rapamycin used in formulation) x100
In Vitro Release Kinetics
In vitro, release, kinetics.,of rapamycin from,rapamycin, loaded GMO,NPs were
performed by dissolving 10 mg of rapamycin loaded GMO NPs in 3 ml PBS (0.01
M, pH 7.4) containing 0.1% Tween -80 . To increase the solubility of rapamycin
in buffer solution as well as to maintain in the sink condition, Tween-80 was

used [27]. The resultant suspension was sonicated for 2 min in an ice bath with
amplitude 30% and divided in three tubes containing 1 ml each. The tubes were
kept in a shaker at 37 ° C at 150 rpm (Wadegati Labequip, India). At particular
time intervals, these tubes were taken out and centrifuged at 13,800 rpm, 25* C
for 10 min (SIGMA 1-15 K, Germany). The supernatants were collected and
lyophilized for 24hr. To the pellet obtained after centrifugation, 1 ml of fresh PBS
containing Tween 80 (0.1%) was added and replaced in the shaker for next
readings. The lyophilized powder was dissolved in 1 ml of acetonitrile for the
dissolution of drug and 20 ul of this solution was injected in the HPCL to quantify
the rapoamycin released with respect to the time intervals.
Cellular uptake study
Cellular uptake study was performed by using 6-coumarin and 6-coumarin loaded
GMO NPs [26]. Briefly, MIA PaCa cells were seeded at a seeding density of 1 x
105 cells per well in a 24 well plate (Corning, NY, USA) and allowed to attach for
24 hr at 37 C in CO2 incubator (Here Cell, Thermo scientific, and Waltham, MA).
The media was replaced with either 100 ng/ml concentration of native 6-
coumarin or 6-coumarin loaded GMO NPS and incubated for different time
periods (lhr, 4 hr and 24.hr) ?t.370C in CO2 incubator After incubation, the cells
were collected and washed twice with cold PBS (0.1 M, pH 7.4). The treated cells
taken for FACS readings to quantify the intracellular 6- coumarin uptake by the
MIA PaCa cells. For demonstrating qualitative cellular uptake the cells were
viewed and imaged under a confocal laser scanning microscope (Leica TCS SP5,
Leica Microsystems GmbH, Germany) equipped with an argon laser using FTTC
filter (Ex 488 nm, Em 525 nm). The images were processed using Leica
Application Suite software.

Sub-Cellular Localisation of GMO NPs by confocal study
We have further studied the in vitro sub-cellular localization of 6-coumarin
loaded GMO NPs in MIA PaCa cells by confocal microscopy. Briefly, Cell
monolayers were cultured in Bioptake® tissue culture plates (Bioptechs, Inc.,
Butler, PA) at a density 10,000 cells per plate and incubated at 370 C in CO2
incubator. Next day, the cell monolayers were treated with 6 -coumarin loaded
GMO NPs (100ng/ml) for different time periods (5, 15, 30 and 60 min). At the
end of the incubation period , cell monolayers were washed thrice with ice cold
PBS (0.1 M, pH 7.4) followed with 10% buffered formaldehyde fixation for 15
min and finally stained with propidium iodide (PI) for 1 hr. The plates were
washed with PBS (0.1 M, pH 7.4 ) and the cells were imaged in confocal laser
scanning microscope (Leica TCS RCS SP5, Leica Microsystems GmbH, Germany)
equipped with an Argon laser using FITC filter (Ex 488nm, Em 525 nm ). The
images were processed using Laica Application suite Software.
Cell Proliferation Assay
Cell viability was evaluated by MTT assay as described previously. Briefly,
different pancreatic cell lines (PANC -1 and MIA PaCa -2 ), leukemia cell line (K-
562) and human colon cancer cell line (HCT -116) were seeded at a density 2000
cells/well in 96 well plates (Corning , NY,USA). Next day, the cells were treated
with different concentration (0.1, 1, 5 10, 50, 100 ng /ml) of either native
rapamaycin dissolved in DMSO or equivalent concentration of rapamaycin loaded
GMO NPs. Concentration of DMSO was kept < 0.1(w/v) so that it will not show
any significant effect on cell proliferation [26]. Medium treated cells and void NPs
treated cells were taken as respective control. After 5 days of incubation, lOul of
MTT reagent was added and incubated for 3 hour in a cell culture incubator at

37 C (Here Cell, Thermo Scientific, Waltman, MA). The intracellular formazon
crystals formed were dissolved in DMSO and the colour intensity was measured
at 540 nm using an ELISA plate reader (Synergy HT, Bio Tek® Instruments Inc.,
Winooski, VT, USA). The antiproliferative effect was measured as a percentage
of cell growth with respective control. The IC50 value was calculated by nonlinear
regression analysis using the sigmoid plot equation.
Mitochondrial depolarization study by using JCI Dye.
The changes in the mitochondrial membrane potential (MMP) were evaluated by
flow cytomatery using JC - 1 dye. In brief, the MIA PaCa cells at a density 2 x
105 were seeded in 6-well plate (Corning, NY, USA). Next day, the cells were
treated with native rapamycin or rapamycin loaded in GMO NPs at a
concentration of 100ng/ml and incubated for 48 hours. After particular time of
incubation the cells were collected by trypsinization and centrifugation (3000
rpm,_ 5 min). The cell pellets were then washed twice with PBS (0.01 M, pH 7.4)
and incubated at 3ul of JC -1 at 370 C for 30 min in dark. Then the cells were
examined for each sample on FL-2 dot plot on Becton Dicknison FACS Caliber.
The data was determined by analyzing 10, 000 gated cells using a FAC scan flow
cytometer. (FACS Caliber: Becton Dickinson, San Jose, CA) and cell Quest ™
software (Becton - Dicnison, San Jose, CA).
Apoptosis study by Flow Cytometry
The induction of apoptosis by native rapamycin and rapamycin loaded GMO NPs
were analyzed by flow cytometry [30].MIA PaCa cells were seeded at density
2x105 cells/ well in 6-well plate (Corning NY, USA) containing 2 ml of qrowth

medium and allowed overnight for attachment at 37 0C . Next day, 2 ml media
containing 50 ng/ml concentration of native rapamycin and equivalent
concentration of rapamycin loaded GMO NPs were added to cells and incubated
for 48 hrs in CO2 incubator (Here cell, Thermo Scientific, Waltham MA). The cells
treated with media and void GMO NP s were taken as controls. After 48 hours,
the cells were washed twice with PBS (0.01 M, pH 7.4) and collected by
trypsinization. The pelleted cells were resuspended with 500ul of 1X binding
buffer, 1 ul Annexin V- FITC and 2ul propidium iodide (Sigma Annexin V- FITC
apoptosis detection kit) and incubated at room temperature in dark for 20 min.
Stained cells were analyzed by FACScan flow cytometer and cell quest software
(FACS Caliber; Becton-Dicknison, San Jose, CA). All experiments were done in
triplicates.
Western Blot Analysis
The molecular mechanism of apoptosis was demonstrated by western blotting
[20]. In brief, MIA PaCa cells (1x 106 cells/ml) were treated with lOOng / ml
rapamycin (both in native and nanoparticulates rapamycin) for 48 hrs. The cells
not treated with drug and treated with void GMO NPs were taken as controls.
After 48 hrs, protein lysates and cell extracts preparation were done by
scrapping the cells. The collected cells then washed with 1 x PBS followed by
detergent lysis [50 mM/L Tris-HCL (pH-8.0 ), 150 mM/L NaCI, 1% NP40, 0.5 %
Na-deoxycholate, 0.1 % SDS, containing protease and phosphatase inhibitor
(Sigma, St Louis, MO) cocktails]. The protein concentration was calculated by the
Pierce BCA protein assay. (Pierce, Rockford, IL). Protein immune detection was
done by electrophoretic transfer of SDS-PAGE separated proteins to PVDF
membrane (PVDF, GE Healthcare) incubated with appropriate primary antibody

recognizing p70S6Kl, 4E-BP1, p-4E-BPl and c-myc (Imgenix corporation, San
Diego, USA), p-Akt and β-actin (Santacruz Microbiology, Inc., Santacruz, CA) and
Bel- 2 (Cell Signaling Technology, Inc, Danver, MA) in 1 :1000 times dilutions for
either overnight at 4° C or 1 h at room temperature with gentle shaking followed
by incubation with goat anti-mouse/rabbit IgG-hoarradish peroidase-conjigated
anti-body (Santacruz Microbiology, Inc., Santacruz, CA) in 1 : 5000 times
dilutions for 40 min at room temperature . The protein of interest was detected
by incubating with Enhanced Chemiluminescence Plus (Amersham Biosciences,
Little Shalfont, and United Kingdom).
In Vivo biocompatibility study of void GMO NPs
In order to justify the nontoxicity nature of void GMO NPs, in vivo toxicity study
was conducted [10]. This study was performed by administering void GMO NPs
with 500 mg/ kg to the BALB/c mice (n=3) via oral gavage needle. Mice were
observed daily for behavioral abnormalities and total body weight were taken in
different time intervals. After 30 days the treated mice were sacrificed and the
histology of major visceral organs were done to observe the histological
abnormalities.
In Vivo Pharmaco kinetics study
The main objective of this experiment was to compare the pharmacokinetic
behaviour of native rapamycin with rapamycijn loaded GMO NPs after oral drug
administration. The experiment was carried out with the permission of
Institutional Animal Ethics Committee of Institute of Life Sciences,
Bhubanesawar, India. For this study male BALB/c mice weighing 20-25 grams

were used. These mice were divided into-two groups (n-3), group 1, mice were
administered native rapamycin dissolved in Milli-Q water with Tween-20 (1% v/v)
and group 2, mice were given rapamycin loaded GMO NPs dissolved in distilled
water at an equivalent dose of 15 mg/kg of native rapamycin by oral gavage
needle. At different time intervals, the peripheral blood from retro-orbital plexus
was collected and the serum was separated. Rapamycin concentration in blood
was measured by HPCL analysis as described earlier [3]. .
Cell Culture
These experiments were performed by taking the above studied cancer cell line
from American Type Culture Collection (Manassas, V A) and were cultured by
using DMEM (PAN BIOTECH GmbH, Aidenbach, Germany) with 10 %fetal bovine
serum (FBS), 1 % L- glutamine and 1 % penicillin -streptomycin. The cells were
maintained at 37 C in a humidified, 5 % CO2 atmosphere in an incubator (Hera
Cells, Thermo Scientific, and Waltham, MA). All other chemicals were procured
from Himedias Laboratories Pvt., Mumbai, India.
Statistical Analysis
Student's t test was used to conduct statistical analysis. Data are expressed as
mean ± standard deviation and values of p < 0.05 were indicative of significant
differences and P< 0.005 was considered very significant differences.

Results
Physicochemical characteristic of rapamycin NPs
. We have successfully prepared aqueous dispersed rapamycin loaded GMO NPs
with an average diameter 88± 6 nm and polydispesity index 0.09 ± 0.02 as
determined by Dynamic Light Scattering measurement (Fig la). The average
zeta potential of the NPs was -31.5 ±3.5, which would certainly increase the
stability of the NPs in dispersion. Studies also supported that particles with such
high negative surface charge are more attracted to the mucosal surface thus
preventing elimination of oral drug formulation through the alimentary canal. For
further confirmation of size distribution and shape of the rapamycin loaded GMO
NPs, TEM and AFM study were performed and the image showed the average
size of particles were - 100nm and - 70 nm respectively. (Fig l.b and c). The
observation further explained the particles were discrete spherical outline with
mono dispersed size distribution. Moreover, the HPLC analysis observed that and
outline with mono dispersed size distribution. Moreover, the HPLC analysis
observed that rapamycin was efficiently loaded in GMO NPs, reaching a high
encapsulated efficiency of 92 ± 3.4 %. Further, FT-IR study was used to
characterize if any chemical interaction occurred in the polymer due to addition
of drug during the nano particle formulation. The FT-IR spectra of rapamycin,
loaded GMO NPs and void GMO NPs are shown in (Fig.2a). The characteristic
peaks due to different functional groups in native rapamycin were appeared at
3418 cm "* due to O-H stretching vibrations, 2875 and 2932 cm "* due to C-H
stretching vibrations, 1718 cm "* corresponds to C=O carbonyl stretching and
1377 cm -1due to-CH bending/ deformation. However, the bands appeared in
void GMO NPs were almost identical to rapamycin loaded GMO NPs in addition to

some peaks due to native rapamycin. The peak at 3418 cm-1 in native rapamycin
due to O-H stretching vibrations of intermolecularly bonded O-H groups was
appeared at 3420 cm -1 in rapamycin loaded GMO NPs, more intensified C-H
stretching vibration bands and C=O carbonyl stretching bands as compared to
void GMO NPs, obtained in nanoparticle surface, indicating the presence of
rapamycin in rapamycin loaded GMO NPs.
The physical status of the drug formulated in the GMO NPs was compared with
the native rapamycin by DSC analysis. The DSC thermogram of native
rapamycin, rapamycin loaded GMO NPs and void GMO NPs were shown in (Fig.
2b), Rapamycin in its natural state exists as crystals, which are characterized by
the high peak at the melting point (180C). However, when encapsulated in the
GMO NPs, the peak at its original melting point disappeared. To understand the
nature of rapamycin in our GMO NPs formulation the XRD study was further
taken into consideration. Then characteristics peaks of native rapamycin
exhibited as shown in (Fig 2c iii), demonstrated the traits of high crystalline
structure. However, there were no characteristics rapamycin peaks were
observed when entrapped in NPs (Fig. 2c ii). This specified the drug was
molecularly dispersed or in amorphous state which favours easy diffusion of drug
molecules through the polymeric matrix, resulting a sustained release of the drug
from NPs . Sustained release of the drug from NPs is an important factor for
achieve successful NP formulations .While observing the in vitro release profile,
we observed a bi-phasic release pattern of entrapped rapamycin from
rapamycin loaded GMO NPs as shown in (Fig.2d). Approximately 32.6 ±1.18 %
of rapamycin was released in 24 hr followed by a sustained release of the drug
about 43.7 ±2.06 % after 14 days. The observed initial burst release could be
due to diffusion of surface absorbed drugs present just beneath the surface of
the NPs, followed by a sustained release of rapamycin entrapped inside.the NPs.
The similar trends of release were also observed by Trickier et al and
dexamethasone in 1 hr respectively.

Sub-cellular localization by confocal microscopy
The GMO NPs formulation was further assessed for cellular uptake studies by
using MIA PaCa cell line as model for pancreatic cells. The quantitative cellular
uptake result demonstrated that 6- coumarin (Fig 3a). The 6-coumarin loaded
GMO NPs showed 2.04, 2.43 and 1.54 times more cellular uptake than native 6-
coumarin after 1 hr, 4 hr and 24 hr of incubation respectively(Fig 3b). Similarly,
in another studies the nuclear co localization of coumarin -6 loaded GMO NPs
were observed qualitatively by confocal microscopy (Fig 4a). On combining the
images of 6-coumarin loaded GMO NPs formulation with the orange-red
propidium iodide stained nuclear material, we have accessed the nuclear co-
localization of our formulation in studied MIA PaCa cells (Fig 4b). At early period
of incubation (up to 30 min) free intracellular green fluorescence of 6-coumarin
were only restricted to cytoplasmic part but at the same time point no nuclear
co-localization of our formulation was noticed. Interestingly, after 1 hr of
treatment green fluoresces of 6-coumarin was monitored in intranuclear region
of MIA Pa Ca cells, suggesting time dependent intracellular localization as well as
nuclear co-localization of our formulation.
In Vitro mitogenic assay
Therapeutic efficiency of drug loaded NPs depends on its uptake, their
intracellular distribution, and more importantly on the dose of the drug that is
released from the internalized NPs inside the cell. So, to investigate the theurapic
efficiency of our formulation, different cancer cells line were treated with void,
native rapamycin and rapamycin loaded GMO NPs at different concentrations for
5 days and cell proliferation was measured by a standard MTT colorimetric assay

Csb values of native rapamycin and rapamycin loaded GMO NPs in different
tumor cells as observed by cytotoxicity assay.

(Fig 5). All the studied cell line showed a typical dose dependent sigmoidal anti
prpliferactive effect. The IC50 value of native and rapamycin loaded GMO NPs, as
obtained from MTT assay in different cell lines were shown in Table 1. These
result demonstrated rapamycin loaded GMO NPs were 1.55, 1.31, 2.27 and 2.76
times more effective than native rapamycin as observed in K 562, HCT-116,
PANC -1 and MIA PaCa-2 cell line respectively. Hence, the obtained results
revealed comparable inhibition of cell proliferation, where rapamycin loaded NPs
were more effective than native in solution by controlling the in vitro cancer cell
growth.

Mitochondrial depolarization study by using JCI dye.
Mitochondria play a pivotal role in apoptosis and loss of MMP (an event in early
apoptosis) is associated with permeability transition. At early stage of apoptosis
the cationic JCI is used to detect MMP as it exhibits potential dependent
accumulation in mitochondria leading to formation of red florescent aggregates
[34]. Here, we have compared the loss of MMP in native rapamycin and
rapamycin loaded GMO NPs treated cells by taking 100ng/ml of drug as shown in
(figure 6.a). We found enhanced apoptosis indicates in rapamycin loaded GMO
NPs i.e 48.77 % than that of native rapamycin 21.25 %. We anticipate that
native rapamycin and rapamycin loaded -GMO. NPs might induce apoptosis
through a mitochondrial pathway whereas the induction of apoptosis is higher in
the case of rapamycin loaded GMO NPs than native rapamycin.
Apoptosis analysis by flow cytometry
The induction of apoptosis by inhibition of m TOR is considered to be one of the
principle mechanisms by which rapamycin have shown tumor regression [5, 26].
Accordingly, we have investigated the ability of rapamycin to induce apoptosis in
pancreatic cancer cells by incubating MIA PaCa cells with equivalent
concentration of 50 ng/ml of native rapamycin and rapamycin loaded GMO NPs
for 48 hours. The results of FACS showed the presence of early apoptotic,
advanced apoptotic and necrotic cell population in all treated cell (fig 6.b).The
fractions of cells that are in early apoptosis were positive for proipidium iodide
and placed in lower right quadrant. The FACS result demonstrated that

rapamycin loaded GMO NPs treated cell showed more number of cells, i.e 13.8
%; in early apoptosis as compared to 1.74 % of cell found in native rapamycin
treated cells. This result suggests that cells treated with rapamycin loaded GMO
NPs were able to cause more apoptosis in MIA PaCa cell line in comparison to
. native drug. In this way, the NPs treated cell showed 7.93 times more apoptosis
than those treated with native rapamycin.
Western Blot analysis
Rapamycin induces apoptosis mostly through inhibition of Akt/mTOR p 70S6K1
pathway, a central pathway of protein translation involved in regulation of cell
proliferation, growth, differentiation and survival. To determine the ability of
rapamycin for promoting apoptosis in MIA Pa Ca cell line, we investigated the
signal transduction pathway induced by rapamycin in MIA Pa Ca cells by western
blot analysis (Fig 7). As evident form fig 7, p-Akt (the key modulator in
upstream pathway), p-p70S6Kl and p-4E BP1 treated cells. In contrast the band
intensity was decreased more in rapamycin loaded GMO NPs treated cells. It is
due to the fact that rapamycin inhibits phosphorylation of Akt, p 70S6K1 and 4E-
BP1 in mTOR signaling pathway. As a result a less activated band of anti-
apoptotic BCL-2 was obtained in NPs as compared to native. However, there is
no significant difference in c-Myc expression on both native rapamycin and
rapamycin loaded GMO NPs treated case. From these above results, it is evident
that our formulation inhibited p-Akt by decreasing the activation level of p-p
70jS6Kl and p-4E BP1 inducing apoptotic signals in m TOR pathway leading to
enhanced cell death.
In Vivo Biocompatibility study of void GMO NPS
The biocompatibility study was conducted by administrating 500mg/kg void GMO

NPs to BALB/c mice by oral gavage needle and kept the animal up to a period of
30 days. As seen in (fig 8.a) despite the relatively large dosage, the mice
receiving void GMO NPs demonstrated no evidence of weight loss and no gross
tissue changes as seen through histological observation. Similarly, no behavioral
changes were observed in the mice during the follow up observation.
In Vivo Pharmakinetic studies
We have compared the bioavailability of rapamycin in native and NPs formulation
by administrating a dose of 15 mg/kg by oral gavage needle to BALB /c mice.
The mean rapamycin concentration in the mice serum n different time intervals
. were exemplified (Fig 8.b). After 2 hr administration of rapamycin. loaded GMO
NPs, the maximum drug concentration availability in serum was 890ng/ml
whereas the availability was 152 ng/ml in native rapamycin. Further, it was found
that the oral bioavailability of rapamycin in rapamycin loaded GMO NPs was high
£-6.8 times) as compared to native rapamycin after 24 hr of administration.
Conclusion
The discussed rapamycin nanoparticulate consist of vitamin-E TPGS and GMO, an
absorption enhancer and bioavailability promoter certainly improved the
therapeutic index of drug loaded nanoparticles compared to native rapamycin.
The observed comprehensible results explained rapamycin loaded GMO NPs was
comparatively more effective than native rapamycin under in vitro condition with
time due to greater cellular uptake, sustained intercellular drug retention and
enhanced antiproliferative effect by inducing apoptosis. Most importantly, the
enhanced cellular internalization and sustained release of entrapped rapamycin
in our formulation results its enhanced systematic bioavailability. Thus, in the

light of the currently still unmet medical need for strategies that focus enhanced
bioavailability of administered drug, the formulated delivery system holds much
promises for the treatment of malignancies in near future.

WE CLAIM:
1. A drug delivery formulation of rapamycin in the form of polymer nanoparticles
for cancer therapy having absorption enhancer & bioavailability promoter
properties.
2. A method for preparing a rapamycin loaded delivery system in the form of
polymer nano particles comprising the step of:
Incorporating rapamycin into a fluid phase of GMO,
Subjecting the said GMO mixture to the step of emulsification with pluronic
solution,
Emulsifying the resultant solution once again with solution of vitamin- E
TPGS;
Lyophilizing the emulsion by freeze drying to produce lyophilized powder having
rapamycin.
3. The method as claimed 2 is a pharmaceutical composition including plurality of
nano particles comprising: a glyceryl mono fatty acid ester, surfactants/stabilizers
. and a cancer therapeutic agent.

4. The pharmaceutical composition according to claim 2, wherein said glycerol
mono fatty acid ester is gyceryl is monooleate (GMO).
5. The method as claimed in claim 2 the stabilizer/ surfactant used in pluronic F-
127 (10 % w/v), wherein the pluronic solution (comprises of hrdrophilic poly
(ethylene oxide) [PEO] and hydrophobic poly (propylene oxide) [PPO)]
. 6. The pharmaceutical composition according to claim 2, wherein another
surfactant used in vitamin-E TPGS (5 % w/v), which is an oral absorption
enhancer.
7. The method as claimed in claim 2, wherein at temperature 40 ° C rapamycin
was incorporated into the fluid phase of GMO.
8. The method as claimed in claim 2, wherein the step of emulsification is
preferred by sonication for 2 min at 30 % amplitude.
9. The method as claimed in claim 2, wherein the lypholization is preferred by
freeze drying method as at -80 °C and <10um mercury pressure.

The present invention relates to a pharmaceutical composition of drug delivery
polymeric nanoparticles for cancer therapy having absorption enhance &
bioavailability promoter properties. The nanoparticles include a glyceryl
monooleate, pluronic F-127, vitamin-E TPGS; and a cancer therapeutic agent,
such as rapamycin (hydrophobic cancer therapeutic agents). Also disclosed are
methods for preparing such nanoparticles and pharmaceutical compositions. The
formulated nanoparticle demonstrating enhanced in vivo pharmacokinetics and in
vitro cellular uptake. As well as, no toxicity and biocompatibility of void particle
at a higher dose of oral administration in animal model.