Description:FILED OF THE INVENTION

The present invention relates to the field of pharmaceutical compositions, and more specifically, to methods for conjugating a cell-penetrating peptide (CPP) named (CR)4 to a composition comprising liposomes. The use of the resulting conjugated CPP-liposome composition for delivering therapeutic agents to targeted cancer cells.

BACKGROUND OF THE INVENTION

Cancer remains a significant global health concern, with conventional treatment methods often facing challenges in achieving optimal efficacy while minimizing harm to healthy tissues. Conventional cancer drug delivery methods face challenges including limited efficacy and harm to healthy tissues. Cell-penetrating peptides (CPPs) offer a promising solution by enabling targeted drug delivery. CPPs are short peptides capable of traversing cellular membranes, facilitating the intracellular delivery of various cargoes, including therapeutic agents. Their ability to penetrate cell membranes makes them attractive candidates for enhancing drug delivery to specific target cells.
In the field of cancer therapeutics, targeting specific molecular markers on cancer cells has been a focus of research to improve treatment outcomes. Prior work in the field has demonstrated the importance of targeting the human epidermal growth factor receptor 3 (HER3) protein in breast cancer therapy, particularly in HER3-positive breast cancer cases. HER3-positive breast cancer is characterized by overexpression of the HER3 protein, which plays a significant role in cancer cell growth and survival. Traditional treatment approaches for HER3-positive breast cancer often involve targeting the HER3 protein using antibodies. However, traditional delivery methods for antibodies can still suffer from limitations in targeting specificity and non-specific distribution within the body.
Doxorubicin, a commonly used antitumor drug, is frequently employed in breast cancer therapy. However, its therapeutic efficacy is hindered by non-specific distribution, leading to adverse effects on healthy tissues. Therefore, there is a significant need for improved methods for treating HER3-positive breast cancer that address the limitations of existing therapies, particularly by overcoming the challenges associated with non-specific drug distribution and improving targeted delivery.

SUMMARY OF THE INVENTION

In an aspect of the present invention, the present invention relates to a liposome composition for targeted drug delivery to breast cancer cells. The liposome composition includes
a) a liposome encapsulating a therapeutic agent and
b) (CR)4 peptide covalently conjugated to the surface of the liposome.
In one embodiment, the therapeutic agent is selected as doxorubicin, anti-cancer agents, and a combination thereof.
In one embodiment, the liposome is surface modified with a DSPE-PEG2000-Malamide-(CR)4 conjugate.
In one embodiment, the DSPE-PEG2000-Malamide-(CR)4 conjugate is prepared by Mal-thiol reaction.
In one embodiment, the (CR)4 in DSPE-PEG2000-Malamide-(CR)4 conjugate represents a sequence of amino acids composed of (cysteine-arginine-cysteine-arginine-cysteine-arginine-cysteine-arginine), serves as a linker for the surface modification of liposomes to enable targeted delivery.
In another aspect of the present invention, a method for preparing a liposome composition for targeted drug delivery to breast cancer cells is disclosed. The method includes a) synthesizing a DSPE-PEG2000-Malamide, b) synthesizing a DSPE-PEG2000-Malamide-(CR)4 conjugate c) preparing a surface-modified liposome (LDM) with the synthesized DSPE-PEG2000-Malamide-(CR)4 conjugate, and d) encapsulating a therapeutic agent in the LDM.
In one embodiment, the therapeutic agent is selected as doxorubicin and anti-cancer agents, and a combination thereof.
In one embodiment, the synthesis of DSPE-PEG2000-Malamide involves reacting amino-PEG2000-DSPE with N-succinimide-3-(N-malamido) propionate (MPS).
In one embodiment, the preparation of the surface-modified liposomes encapsulating anti-cancer drug by remote loading technique.
These elements, together with the other aspects of the present invention and various features are pointed out with particularity in the claims annexed hereto and form a part of the present invention. For a better understanding of the present invention, its operating advantages, and the specified object attained by its uses, reference should be made to the accompanying drawings and descriptive matter in which there are illustrated exemplary embodiments of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 illustrate the pathway of DOX-loaded surface modified liposomes (LDM) from extracellular to intracellular environment;
Figure 2 illustrates the mechanism of action of Doxorubicin (DOX) in various cell organelles;
Figure 3 illustrates the diagram illustrating the synthesis process of DSPE-PEG2000-Malamide;
Figure 4 illustrates FTIR peaks of DSPE-PEG2000-Malamide;
Figure 5 illustrates 1H-NMR peaks of DSPE-PEG2000-Malamide;
Figure 6 illustrates 13C-NMR peaks of DSPE-PEG2000-Malamide;
Figure 7 illustrates a diagram illustrating the synthesis process of DSPE-PEG2000-Mal-(CR)4/Conjugate;
Fig 8 illustrates the TLC of the Conjugate;
Figure 9 illustrates FTIR peaks of the Conjugate;
Figure 10 illustrates 1H-NMR peaks of the Conjugate;
Figure 11 illustrates 13C-NMR peaks of Conjugate;
Figure 12 illustrates Mass spectroscopy of Conjugate;
Figure 13 illustrates Schematic representation of the preparation of liposomes by thin-film hydration method;
Figure 14 illustrates the fabricated CPP modifies liposomes ((CR)4 -Lip-DOX) involve various types of intelligences in tumor cells or tissues;
Figure 15 illustrates 3D-response plots (a-c) and contour plots (d-f) showing the effects of molar ratio HSPC, the molar concentration of DSPE-PEG2000, and conjugate on Particle Size;
Figure 16 illustrates 3D-response plots (a-c) and contour plots (d-f) showing the effects of molar ratio HSPC, the molar concentration of DSPE-PEG2000, and conjugate on Zeta potential;
Figure 17 illustrates 3D-response plots (a-c) and contour plots (d-f) showing the effects of molar ratio HSPC, molar concentration of DSPE-PEG2000, and conjugate on Entrapment Efficiency;
Figure 18 illustrates the overlay graph of the optimized formulation obtained after numerical and graphical optimization indicating the composition of the optimized formulation;
Figure 19 illustrates FTIR spectra of Doxorubicin (a), cholesterol (b), HSPC (c), mPEG2000-DSPE (d), and a physical mixture of excipients and DOX (e)
Figure 20 illustrates FTIR Peaks of blank liposomes (L)
Figure 21 illustrates 1H -NMR of blank liposomes (L)
Figure 22 illustrates FTIR peaks of conjugated liposomes LDM
Figure 23 illustrates 1H-NMR of Conjugated Liposomes LDM
Figure 24 illustrates Differential Scanning Calorimetry (DSC) and thermogram (TG) of Doxorubicin (a), cholesterol (b), HSPC (c), mPEG2000-DSPE (d), and physical mixture of excipients and DOX (e);
Figure 25 illustrates DSC thermogram peaks of blank liposomes (L);
Figure 26 illustrates DSC thermogram peaks of conjugated liposomes LDM peaks at 80.5ºC and 280ºC;
Figure 27 illustrates (a) Mean Particle Size (158.8 ± 1.69) and PDI (0.274) of DOX-loaded liposomes (LD); (b) Mean Particle Size (118.5 ± 1.28) and PDI (0.291) of DOX-loaded surface modified liposomes (LDM);
Figure 28 illustrates Zeta potential of a) DOX-loaded liposomes (LD); b) DOX-loaded surface modified liposomes (LDM);
Figure 29 illustrates Light microscope image of (a) LD (b) LDM;
Figure 30 illustrates Transmission electronic microscope (TEM) images of a) DOX-loaded liposomes (LD); b) DOX-loaded surface modified liposomes (LDM);
Figure 31 illustrates Percentage cumulative drug release of formulations D, LD, LDM at pH 6.8
Figure 32 illustrates SRB assay for cytotoxicity studies
Figure 33 illustrates Graphical representation of % Growth inhibition and IC50 of D (plain DOX solution); blank liposomes (L); DOX-loaded liposomes (LD) and LDM (DOX-loaded surface modified liposomes)
Figure 34 illustrates Cytotoxicity studies in MCF-7 cell line a) ADR b) Control, c) Formulation code D, d) Formulation code L, e) Formulation code LD, f) Formulation code LDM
Figure 35 illustrates Detection of intracellular ROS production in MCF-7 cells lines.
The cells were treated with a) Negative Control, b) H2O2 (0.05%), c) LDM (2.5 µM), d) LDM (5.0 µM), e) LDM (7.5 µM) for 48 h. The fluorescence intensity of DCFDA in the case of LDM (5.0 µM), e) LDM (7.5 µM) is significantly high. H2DCFDA fluorescent at Ex/Em 495/529nm, Green dye; Fluorescence intensity is directly proportional to ROS levels;
Figure 36 illustrates the Effect of LDM on Nuclear Morphology (DAPI Staining) in MCF-7
The cells were treated to DOX and various concentration of formulation LDM for 48 h., then stained with DAPI to observe if any morphological changes occurred.
(a) Untreated cells (negative control) had normal nuclear morphology, (b) treated cells with 1.9 µM DOX was showed formation of typical apoptotic bodies, (c) LDM (2.5 µM) treated cells caused minor nuclear morphological change, (d) and (e) cells treated with LDM (5.0 and 7.5 µM), revealed morphological alterations were noticeable. DAPI fluroscent at Ex/Em 350/465 nm, Blue dye. The arrow pointed to the formation of apoptotic bodies;
Figure 37 illustrates Detection of loss of MMP in MCF-7 cells. The cells were treated with formulations for 48 h; (a) Untreated cells (negative control), (b) the fluorescence intensity of rhodamine-123 in the case of free 1.9 µM DOX, (c) LDM (2.5 µM), (d) LDM (5.0), (e) LDM (7.5 µM). Rhodamine-123 fluroscent at Ex/Em 480/530 nm, Red dye;
Figure 38 illustrates Demonstration of coagulation assay by Prothrombin Time (PT) ; Activated Partial Thromboplastin Time (APTT) LD & LDM nano formulations, (Mean+ SD, n=3).
Figure 39 illustrates Hemolytic activity of the different liposomal formulations (D, LD, LDM);
Figure 40 illustrates Image A & B shows hemolysis analysis of optimized DOX-loaded surface modified liposomes (LDM) at various concentration of blood (0.2-1.0 mg/ml). Image C (were a=positive control, b=negative control, c= 0.2 mg/ml, d=0.4, e=0.6, f= 0.8 mg/ml and g= 1.0 mg/ml of LDM) demonstrates microscopic analysis of blood smear slide stained with 1 % w/v Field’s reagent and visualized at 60X and images captured in Trans mode at 60X magnification using light microscope (Evos, Japan). The data exhibit no rupture of red blood cells in LDM treated blood, (Mean+ SD, n=3);
Figure 41 illustrates Human blood parameters panel data control, plain DOX solution (D), blank liposomes (L), DOX-loaded liposomes (LD), DOX-loaded surface modified liposomes (LDM), (Mean+ SD, n=3). a) RBC count; b) WBC count; c) Lymphocytes count; d) HGB (Hemoglobin); e) HCT (hematocrit); f) PLT (Platelets);
Figure 42 illustrates the percent dose recovered in different organs after i.v. administration of various formulations;
Figure 43 illustrates In vivo evaluation (a) Comparison of % change in tumor volume of different groups (b) % tumor burden of free drug and drug loaded formulations in tumors; (c) Relative size of the tumor harvested from the animals;
Figure 44 illustrates Histopathological sections of (A) liver; (B) kidney; (C) heart; (D) brain; (E) tumor after the treatment of (a) control, (b) D, (c) L, (d) LD, (e) LDM.
Figure 45 illustrates Histogram showing effect of Storage conditions on av. vesicle size and % Residual drug content of LDM formulations
Like reference numerals refer to like parts throughout the description of several views of the drawing.

DETAILED DESCRIPTION OF INVENTION

Embodiments are provided so as to thoroughly and fully convey the scope of the present invention to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present invention. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present invention. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

The terminology used, in the present invention, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present invention. As used in the present invention, the forms "a," "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," "including," and "having," are open-ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units, and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present invention is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.

In an aspect of the present invention, the present invention relates to a liposome composition for targeted drug delivery to breast cancer cells. The liposome composition includes a) a liposome encapsulating a therapeutic agent and b) (CR)4 peptide covalently conjugated to the surface of the liposome.

In one embodiment, the therapeutic agent is selected as doxorubicin and anti-cancer agents.
In one embodiment, the liposomes are surface-modified with a DSPE-PEG2000-Malamide-(CR)4 conjugate.
In one embodiment, the DSPE-PEG2000-Malamide-(CR)4 conjugate is prepared by Mal-thiol reaction.

In one embodiment, the (CR)4 in DSPE-PEG2000-Malamide-(CR)4 conjugate represents a sequence of amino acids composed of (cysteine-arginine-cysteine-arginine-cysteine-arginine-cysteine-arginine), serves as a linker for the surface modification of liposomes to enable targeted delivery.
In another aspect of the present invention, a method for preparing a liposome composition for targeted drug delivery to breast cancer cells is disclosed. The method includes a) synthesizing a DSPE-PEG2000-Malamide, b) synthesizing a DSPE-PEG2000-Malamide-(CR)4 conjugate c) preparing a surface-modified liposome (LDM) with the synthesized DSPE-PEG2000-Malamide-(CR)4 conjugate, and d) encapsulating a therapeutic agent in the LDM.

In one embodiment, the therapeutic agent is selected as doxorubicin and anti-cancer agents.

In one embodiment, the synthesis of DSPE-PEG2000-Malamide involves reacting amino-PEG2000-DSPE with N-succinimide-3-(N-malamido) propionate (MPS).

In one embodiment, the preparation of the surface-modified liposomes encapsulating anti-cancer drug by remote loading technique.
Data are shown in Figure 1,2.

Method of preparation

The invention discloses surface-modified liposomes by conjugating peptides onto liposomal surfaces. This invention includes synthesizing DSPE-PEG2000-Malamide, a molecular bridge connecting the peptide to the liposomal membrane. Next, this linker molecule with the chosen peptide through its (CR)4 motif, forging a strong bond by synthesis of DSPE-PEG2000- Malamide-(CR)4 Conjugate. Utilizing the peptide-conjugate, it proceeded to prepare two separate formulations: surface-modified liposomes (LDM) incorporating the peptide and DOX-loaded liposomes (LD) without conjugation.
Synthesis of DSPE-PEG2000-Malamide

For the synthesis of DSPE-PEG2000-Malamide 500mg of amino-PEG2000-DSPE, which is equivalent to 0.18 mM. Additionally, 62.6 mg of N-succinimide-3-(N-malamido)propionate (MPS) equivalent to 0.24 mM is utilized simultaneously. This yielded a molar ratio of 0.75:1 for amino-PEG2000-DSPE to MPS. The reaction took place by dissolving amino-PEG2000-DSPE in dichloromethane (CH2Cl2) and MPS in dimethylformamide (DMF), with the addition of triethylamine. After a 15-minute reaction period, confirmation is obtained through thin-layer chromatography (TLC) using UV light, employing a solvent mixture of chloroform, methanol, and water (90:18:2). The product mixture is then purified via passage through a Sephadex G-50 column, resulting in the isolation of Mal-PEG2000-DSPE as a pure white solid under reduced pressure. Further confirmation of the product's structure was achieved through FTIR and NMR analysis of the reaction mixture. Data are shown in Table 1, Figure 3, 4, 5, 6.

Table 1: FTIR peaks and their corresponding functional groups for DSPE-PEG2000-MAL

Peak Position (cm-1) Functional Group
3477.88 C-H stretching (aliphatic chains)
2922.64 C-H stretching (methylene, -CH2-)
1657.72 C=O stretching (ester carbonyl group)
1385.09 C-H bending (methyl, -CH3)
1253.64 C-O stretching (ether linkage)
1090.27 C-O-C stretching (ether linkage)

Synthesis of DSPE-PEG2000-Malamide-(CR)4 Conjugate

To synthesize the DSPE-PEG2000-Malamide-(CR)4 conjugate, start by dissolving 2.5 mg of the peptide in 4 mL of HEPS Buffer. Next, hydrate the dried lipid film containing 20 mg of DSPE- PEG2000-Mal in 1 mL of HEPS buffer, and carefully add it dropwise to the peptide solution with gentle agitation at room temperature. Stir the resulting solution for 48 hours under N2 protection to facilitate the conjugation process. Afterward, incubated the solution with L-cysteine for an additional 4 hours to react with any remaining maleimide groups. To remove any excess peptide and L-cysteine, dialyze the reaction mixture using a membrane with a molecular weight cutoff of 3500 Da against distilled water for 48 hours. Finally, lyophilized the solution and stored it at - 20°C. Verification of successful conjugation can be performed through FTIR and 1H-NMR analysis of the resulting product. Data are shown in Table 2, Figure 7-12.

Table 2: FTIR peaks and functional groups for DSPE-PEG2000-MAL-(CR)4

Peak Position (cm-1) Functional Group
2979.09 Thiol (S-H)
1710.05 Maleimide (C=C)
1636.82 C=O, carbonyl stretch (Peptide bonds)
1448.45 C=O-NH, amide II (Peptide bonds)
1365.64 C=O, ester groups in the PEG linker

Preparation of Surface Modified Liposomes (LDM)
Liposomes were prepared using a remote loading technique reported by Wang et al., 2015 [32] employing ammonium sulfate. The liposomes were formed by dissolving HSPC, CHL, and DSPE- PEG2000 in chloroform and methanol, adding DSPE-PEG2000-Malamide-(CR)4 conjugate, evaporating the solvent, and hydrating the lipid film with ammonium sulfate. The mixture was sonicated and dialyzed to remove unencapsulated ammonium sulfate. DOX-loaded liposomes were prepared by mixing the blank liposome suspension with DOX solution, incubating at 40°C, and removing unencapsulated DOX by centrifugation and filtration by Sephadex G-25 mini-columns as shown in figure 3. Liposomes were modified with DSPE-PEG2000-(CR)4 and DSPE- PEG2000 to enhance cellular uptake and serum stability, respectively. Longer cysteine (Cys)- cleavable PEG2000 was incorporated to shield (CR)4 during circulation and switch on its function after tumor accumulation. Data are shown in Figure 13-14.
Optimization

The optimization studies were carried out employing three-factors-two-level (2×3) BBD as shown in Table 3. Table 4 enlists the layout of a total of 17 runs planned as per BBD for the ensuing experimental studies, using the chosen CMAs at three different levels. The different CQAs like mean particle size (nm), zeta potential (mV), and % EE of the developed formulation(s) were evaluated as per the standard procedures briefly described in the respective sections. Mathematical modeling was subsequently carried out, and various statistical parameters were evaluated to ratify the best model fitting of the experimental data. The interrelationship(s) among the selected CMAs and CQAs were analyzed by employing response surface methodology. Data are shown in Table 3, 4, 5 and Figure 15-18.

Y= ß0 + ß1A + ß2B + ß3C + ß4AB + ß5AC + ß6BC + ß7A² + ß8B² + ß9C² (Eq. 1)
Table 3: Factors and their levels as per Box- Behnken designs (BBD) for optimization of surface-modified liposomes (LDM)

Factors (Independent Variables) Levels
Low (-1) Medium (0) High (+1)
X1: molar conc. of Soya PC (µM) 5.8 6.1 6.4
X2: molar conc. of DSPE-PEG2000 (µM) 0.1 0.25 0.5
X3: molar conc. of Conjugate (µM) 0.05 0.1 0.2
Cholesterol Constant
Doxorubicin Constant
Response (Dependent Variables) Constraints
Y1: Vesicle Size (nm) Minimum
Y2: Zeta Potential (mV) In Range
Y3: Entrapment Efficiency (%) Maximum

Table 4: BBD Design Matrix and experimental results of various formulation and process variables

Run Factor
X1 Factor
X2 Factor
X3 Response
Y1 Response
Y2 Response
Y3
Soya PC (µM) DSPE- PEG2000 (µM) Conjugate (µM) Vesicle Size (nm) Zeta Potential (mV) Entrapment Efficiency (%)
1 6.1 0.3 0.125 111.6 9.04 62.5
2 6.1 0.1 0.200 189.4 9.4 69.62
3 5.8 0.1 0.125 124.9 10.2 69.36
4 6.4 0.5 0.125 176.9 6.3 68.2
5 5.8 0.3 0.200 138.6 11.1 69.52
6 5.8 0.5 0.125 225.2 8.91 67.92
7 6.4 0.3 0.050 123.3 4.41 65.15
8 6.1 0.5 0.050 223.9 3.5 64.51
9 6.1 0.1 0.050 121.6 4 59.73
10 6.1 0.3 0.125 109.5 9.2 61.5
11 6.1 0.3 0.125 110.5 9.4 63.1
12 6.1 0.3 0.125 112.5 9 62.5
13 6.4 0.3 0.200 178.9 14.3 69.5
14 6.1 0.5 0.200 182.6 6.9 65.6
15 6.1 0.3 0.125 111.5 9.8 61.3
16 5.8 0.3 0.050 167.9 13.1 65.18
17 6.4 0.1 0.125 168.2 7.18 66.29

Table 5: Polynomial coefficients of different quadratic model terms

Factors Coefficient code Particle Size Zeta Potential %EE
ß0 111.12 9.29 62.18
Soya PC X1 ß1 -1.16 -1.39 -0.355
DSPE-PEG2000 X2 ß2 25.56 -0.6462 0.1538
DSPE-PEG2000-(CR)4 X3 ß3 6.6 2.09 2.46
Soya PC * DSPE-PEG2000 X1X2 ß4 -22.9 0.1025 0.8375
Soya PC * Conjugate X1X3 ß5 21.23 2.97 0.0025
DSPE-PEG2000 * Conjugate X2X3 ß6 -27.28 -0.5 -2.2
Soya PC² X1² ß7 17.74 1.82 4.12
DSPE-PEG2000 ² X2² ß8 44.94 -2.96 1.64
Conjugate² X3² ß9 23.32 -0.379 1.04

Conjugation efficiency

The conjugation efficiency of (CR)4 with surface modified liposomes (LDM) was determined using the Folin-Ciocalteu reagent. The amount of unconjugated (CR)4 in the supernatant was determined by a UV–visible spectrophotometer (Shimadzu® 1800, Japan) at 750 nm. The percentage conjugation was calculated using Eq. (1).
Percentage congugation =(Initial added (CR)4-recovered (CR)4)/(intial added (CR)4) X100 ……………(Eq. 2)

Characterization of Surface Modified Liposome Formulations

FTIR Analysis

Fourier-transform infrared spectroscopy (FTIR) (Bruker FTIR 8400S ALPHA) was performed for DOX, each excipient individually, a physical mixture of DOX, cholesterol, HSPC, and mPEG2000- DSPE, blank liposomes and LDM. Samples and excipients were vacuum-dried for 12 hours before IR analysis. Dried samples were placed on a sample platform, and spectra were captured within the range of 4000 to 400 cm-1, aligning with the official IR spectrum reported in the monograph. Data are shown in Table 6, 7 Figure 19-23.

Table 6: FTIR peaks and their corresponding functional groups for blank liposomes (L)

Wavenumber (cm-1) Functional Group
2800 - 3000 C-H stretching (aliphatic chains)
1740 C=O stretching (ester carbonyl group)
1650 C=O stretching (amide carbonyl group in PC)
1575 C=C stretching (unsaturation in cholesterol)
1460 C-H bending (methylene, -CH2-)
1100 - 1150 C-O stretching (ether linkage)

Table 7: FTIR peaks and their functional groups for DOX-loaded surface modified liposomes (LDM) with DSPE-PEG2000-MAL-(CR)4

Wavenumber (cm-1) Functional Group
2800 - 3000 C-H stretching (aliphatic chains)
1740 C=O stretching (ester carbonyl group)
1650 C=O stretching (amide carbonyl group in PC)
1575 C=C stretching (unsaturation in cholesterol)
1550 Amide I band (peptide backbone, CR8)
1460 C-H bending (methylene, -CH2-)
1350 Amide III band (CR8)
1100 - 1150 C-O stretching (ether linkage)

3.2 Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed on DOX, all individual excipients, a physical mixture of DOX, cholesterol, HSPC, and mPEG2000-DSPE, and SMPLs using a DSC 60 (Shimadzu, Japan) operated with TA Q series Advantage software (Universal analysis 2000). The device was calibrated with high-purity indium (99.9%). Briefly, about 2-4 mg of each sample was placed in an air-tight sealed aluminum pan and scanned at a rate of 10 °C/min under a dry, inert atmosphere. Data are shown in Figure 24-26.

3.3 Particle Size Distribution, Polydispersity, and Surface Charge of Vesicles

The mean particle size (nm), polydispersity index (PDI), and zeta potential (mV) of LDM were measured employing Zeta sizer Nano ZS (Malvern Instruments Ltd, UK). Data are shown in Figure 27-28.

3.4 Encapsulation Efficiency

The encapsulation efficiency of the prepared carrier system was determined using an indirect method described previously. Briefly, 2 mL of the LDM dispersion was centrifuged at 10,000 rpm (~11,200g) for 10 minutes. The unentrapped drug in the supernatant was extracted with methanol and quantified by UV spectrophotometry (Thermo Scientific Multiskan GO UV/Vis microplate spectrophotometer, Japan) at a wavelength of 480 nm after adjusting the volume. The results are shown in Table 2.

3.5 Transmission Electron Microscopy

The size of the optimized formulation was evaluated using a transmission electron microscope (Jeol JEM1230, Tokyo, Japan). A small drop of the sample was placed on a carbon-coated grid and negatively stained with 1% phosphotungstic acid. The sample was then air-dried and photographed at a suitable magnification. Data are shown in Figure 29-30.

3.6 In Vitro Drug Release Kinetic Modelling Studies

In vitro release of drug from Plain DOX solution (D), DOX-loaded liposomes (LD), and DOX- loaded surface modified liposomes (LDM) were evaluated using the dialysis method. Briefly, 5 ml liposomal suspension was placed in a presoaked dialysis bag (molecular weight cut off 6000-8000 Daltons). The drug release was determined in media i.e. PB (pH 6.8): methanol (7:3) containing 0.1% v/v Tween 80. The release experiments were started by placing the dialysis bag into 50ml of release media with continuous shaking at 300 rpm at 37±1°C. Samples (0.5 mL) were withdrawn at predetermined time intervals from release media over 72 hours period and replaced with the same amount of fresh media. The concentration of DOX was determined by HPLC at 480 nm. The percentage of cumulative DOX released from liposomes was calculated using Eq (3). The experiment was performed in triplicate. Different kinetic models, such as zero-order, first-order, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas release kinetic models, were also used to analyze the release data and identify the release pattern results are shown in Table 6. Data are shown in Table 8 and Figure 31.

Percentage cumulative DOX released =(Cumulative amount of DOX obtained at time)/(Total amount of DOX in liposomes) X100
(Eq-3)

Table 8: Release Kinetics Model of formulations at pH 6.8

Release Kinetics Model Formulation Code
D LD LDM
Zero-order F=k0*t k0 26.729 0.832 1.110
R² adj 0.8867 0.7463 0.8013
AIC 35.0850 82.3747 87.1500
MSC 1.4452 1.0088 1.2769
First-order
F=100*[1-Exp(-k1*t)] k1 0.545 0.012 0.019
R² adj 0.9516 0.8461 0.9274
AIC 30.8341 76.3759 75.0691
MSC 2.2954 1.5087 2.2837
Higuchi F=kH*t0.5 kH 45.085 6.070 8.045
R² adj 0.8962 0.9928 0.9957
AIC 34.6479 39.5874 41.2445
MSC 1.5326 4.5744 5.1024
Korsmeyer-Peppas F=kKP*tn kKP 37.097 7.412 8.406
R² adj 0.9206 0.9977 0.9955
AIC 33.8712 27.0507 42.6451
MSC 1.6880 5.6192 4.9857
Hixson-Crowell
F=100*[1-(1-kHC*t)3] kHC 0.156 0.003 0.005
R² adj 0.9695 0.8175 0.8968
AIC 28.5179 78.4243 79.2830
MSC 2.7586 1.3380 1.9325

Cell Line Studies

4.1 Cell Cultures

Cancer cells such as A549 (lung), MCF-7 (breast), HCT-116 (colon), and MiaPaca-2 (pancreas) were all cultured in a 96-well plate. DMEM (Sigma D5523) was used to culture the MCF-7 and MiaPaca-2 cell lines, while RPMI 1640 (Sigma R6504) was used to culture the A549, and HCT- 116, cell lines. The cells were seeded (6000 cells/well) in 96-well plates and incubated at 37?C with 5% CO2 and 95% relative humidity for 24 hours in a cell-culture incubator.
4.2 In vitro Cytotoxicity Screening

In vitro, cytotoxicity of plain DOX solution (D), blank liposomes (L), DOX-loaded liposomes (LD), and DOX-loaded surface modified liposomes (LDM) was performed on various cell lines using sulforhodamine B (SRB) assay. The samples were diluted and treated with drug concentrations of 1, 5, 10, 20 µM. The experiment was terminated after 48 hours by adding 50 µL of cold TCA to the plates, then incubated for 1 hour at 4-8?C, washed 3-4 times with distilled water, and dried. 100 µL SRB (0.4% w/v) dye was added to each well and incubated for 1 hour at 25±2 ?C. After 1 h, the plate was washed with 1% v/v acetic acid to remove the unbound dye and allowed to dry. Finally, 100 µL of Tris HCl was added to each well, then the absorbance at 540 nm using a multi-well plate reader (TECAN® INFINITE M NANO). The percentage inhibition was calculated using Eq. (4). The IC50 was determined using Graph Pad Prism 8.1. (Graph Pad Software Inc, USA). All the assays were performed in triplicate. The data were represented as the mean and standard deviation of three experiments.
%growth inhibition =100-(absorbance of treated cells)/(absorbance of untreated cells) (Eq. 4)

In all cancer cell lines, the lowest IC50 of DOX-loaded surface modified liposomes (LDM) was obtained in MCF-7 cells. Hence, MCF-7 cells were selected for further mechanistic investigation. Results are shown in Table 6. Data are shown in Table 9 and Figure 32-34.
Table 9: In vitro cytotoxicity of plain DOX solution (D), L, LD and DOX-loaded surface modified liposomes (LDM) against a panel of NCI-60 Human cancer cell lines by SRB assay at 48 hours (mean ± SD, n = 3).

Tissues Lung Breast Colorectal Pancreatic
Cell Lines A549 MCF-7 HCT-116 MIAPaCa-2
Formulations IC50 (µM) (48 hour Experiment)
D 12.2 3.5 10.1 19.4
L 41 24.6 67 44.2
LD 31.2 28.6 49.2 52.1
LDM 20 4.9 22.1 20.4
4.3 Estimation of ROS generation
Detection of intracellular ROS production in MCF-7 cells lines.
The cells were treated with a) Negative Control, b) H2O2 (0.05%), c) LDM (2.5 µM), d) LDM (5.0 µM), e) LDM (7.5 µM) for 48 h. The fluorescence intensity of DCF-DA in the case of LDM (5.0 µM), e) LDM (7.5 µM) is significantly high. H2DCFDA fluroscent at Ex/Em 495/529nm, Green dye; Fluroscence intensity is directly proportional to ROS levels. Data are shown in Figure 35.

4.4 Apoptosis assay through DAPI staining
Effect of LDM on Nuclear Morphology (DAPI Staining) in MCF-7
The cells were treated to DOX and various concentration of formulation LDM for 48 h., then stained with DAPI to observe if any morphological changes occurred.
(a) Untreated cells (negative control) had normal nuclear morphology, (b) treated cells with 1.9 µM DOX was showed formation of typical apoptotic bodies, (c) LDM (2.5 µM) treated cells caused minor nuclear morphological change, (d) and (e) cells treated with LDM (5.0 and 7.5 µM), revealed morphological alterations were noticeable.
The arrow pointed to the formation of apoptotic bodies.
DAPI fluroscent at Ex/Em 350/465 nm, Blue dye. Data are shown in Figure 36.

4.5. Loss of mitochondrial membrane potential (??m)
Detection of loss of MMP in MCF-7 cells. The cells were treated with formulations for 48 h.
(a) Untreated cells (negative control), (b) the fluorescence intensity of rhodamine-123 in the case of free 1.9 µM DOX, (c) LDM (2.5 µM), (d) LDM (5.0), (e) LDM (7.5 µM)
Rhodamine-123 fluroscent at Ex/Em 480/530 nm, Red dye. Data are shown in Figure 37.

5. Coagulation Assay by Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) Assessment
The PT and APTT assay was employed for the determination of coagulation effect of optimized DOX-loaded liposomes (LD), DOX-loaded surface modified liposomes (LDM) formulation. Fresh blood was collected in 10 ml ACD containing tubes and was centrifuged at 5000 rpm for 5 minutes at 25ºC to obtain platelets poor plasma (PPP). 1000 µl of PPP was mixed with 0.1 µl of sample and incubated at 37ºC for 25 minutes. After incubation the PT and APTT were assessed by employing coagulation analyzer regent kit (CK Prest and Fibriprest, Diagnostica Satgo, France). Data are shown in Table 10 Figure 38.
Table 10: Illustration of coagulation assay by Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) of LD & LDM nano formulations, (Mean+ SD, n=3).

S. No. Formulation Code Prothrombin Time (PT) (sec) Activated Partial Thromboplastin Time (APTT) (sec)
1. LD 19.2 18.6
2. LDM 22.3 21.2

Hemolytic Toxicity

The RBC suspension was obtained using the method reported in literatures. Human blood was collected in HiAnticlot blood collecting vials (Hi media, India) with heparin as an anticoagulant and centrifuged at 3000 rpm for 15 minutes. RBCs were collected from the bottom and washed with normal saline (0.9 percent w/v) until a clear, colorless supernatant was formed above the cell mass. The cells were resuspended in saline solution. The RBC suspension prepared by the above method was then employed in the hemolytic study. In control, 5 mL of normal saline was added to 1 mL of RBC suspension. Similarly, plain DOX solution (D) and formulations (L, LD, LDM) were taken into separate tubes along with 1 mL RBC suspension in such an amount that the final concentration of drugs and formulation were equivalent in all cases. The tubes were centrifuged for 15 minutes at 3000 rpm after being allowed to stand for half an hour with gentle intermittent shaking. The supernatants were obtained and diluted with an equal volume of normal saline, and optical density (OD) was measured at 540 nm. The same procedure was used for all formulations. The percent hemolysis was calculated for each sample by applying the following equation (Eq 5). Data are shown in Figure 39.
%Hemolysis =((OD (test)-OD (negative control)))/((OD (positive control)-OD (negative control))) X 100 …………..(Eq. 5)

Haemolysis Assay

The hemolysis evaluation was carried out for optimized DOX-loaded surface modified liposomes (LDM) using fresh human blood. Briefly, 2 ml of ACD solution (acid citrate dextrose) was mixed with 2 ml of blood sample and incubated for 1-2 h at 37?C (100 µL of blood sample concentration ranging from 0.2 to 1.0 mg/ml). The incubated samples were centrifuged at 5000 rpm for 5 minutes to obtain plasma. The obtained plasma was then mixed with 1 µl of 0.01% sodium bicarbonate solution. The samples were then scanned at 450, 380 and 415 nm. The plasma hemoglobin was measured by employing the equation:
Plasma Hb = {(2A415)-[A380+A450] x76.25}

The values obtained for samples were compared with that of normal saline as negative control and triton as positive control. Each concentration was evaluated in triplicate. For the smear slide preparation of blood and evaluation of optimized DOX-loaded surface modified liposomes (LDM), push (wedge) and coverslip technique were employed. In this methodology a drop of fresh blood was placed on the slide at one end with the help of pipette. The smear was prepared by using spreader slide placed on the blood drop perpendicular at an angle of 45? which spread the blood evenly on the base slide avoiding any tailing and uneven width. The smear was then carefully air dried for 30 minutes for proper fixation. The fixed smear was then stained with Field stain (field’s stain B (eosin) and field’s stain A (methylene blue)) and floored for about 15 minutes for cell picking and finally washed with water and observed under the fluorescent microscope at 40x resolution lens (Evos, Japan). Data are shown in Figure 40-41.

In vivo assessment

6.1 DMBA Tumor induction
The animals (female Balb/c mice) were randomly divided into 4 groups. 7,12- dimethylbenz(a)anthracene (DMBA) (a carcinogen) was used to develop an animal model of breast cancer. DMBA-induced breast cancer was developed by subcutaneous injection of DMBA (10 mg/kg BW (body weight)) into the mammary glands of animals. Repeated injections of DMBA solution were injected on two bottom nipples (the right and left regions), divided into two doses. In this experiment, at six weeks of age, 100µl of DMBA (1 mg/ml in gingelly oil) was subcutaneously injected one time per week for six weeks. Each mice from all 4 groups received 100µl of DMBA. Mice were examined daily for the appearance of tumors by palpitation, and the first day of tumor detection was recorded.
6.2 Biodistribution Study

The in vivo distribution profile of developed liposomes was assessed in DMBA-induced breast tumor-bearing female Balb/c mice (22-25 g). All tumor-bearing mice were divided into four groups. They were fasted overnight before the administration of dose but allowed free access to water. The first group was administered with the Plain DOX solution, and the second, third, and fourth groups were administered with blank liposomes (L), DOX-loaded liposomes (LD), and DOX-loaded surface modified liposomes (LDM), respectively. All freeze-dried formulations were dispersed in deionized water (500 µl) and administered through tail vein injection at a DOX dose of 5 mg/kg of body weight. At a definite time, interval, the Balb/c mice from each group were sacrificed, and blood samples were collected via cardiac puncture in heparinized tubes. Organs like kidney, spleen, liver, and tumor were isolated, and washed with ringer’s solution to remove any adhered debris, and dried using tissue paper. The liver, kidney, heart, brain and tumor were weighed and cut into small pieces. Whole organ was homogenized with PBS (pH 7.4) using a tissue homogenizer (MAC Micro Tissue Homogenizer, New Delhi, India) at 4ºC. The 150 µl of tissue homogenate was mixed with an equal volume of acetonitrile. Then, the samples were mixed by vortexing for 30 seconds and centrifuged for 10 minutes at 5000 rpm. The fatty layer was discarded, and the supernatant was collected. The clear supernatant was diluted with PBS (pH 7.4) and filtered through a 0.45 µm syringe filter and the amount of DOX in various organs was determined by HPLC method. Additionally, the amount of drug in blood was also determined by centrifugation (5000 rpm for 10 min) of the collected blood. Then the serum and blood cells were separated, 10 mL of serum was mixed with an equal volume of cold acetonitrile. The samples were mixed by vortexing for 30 seconds and centrifuging for 10 minutes at 5000 rpm. The clear supernatant was filtered through a 0.45 µm filter and analyzed for drug content by HPLC. Data are shown in Figure 42.
6.3 In vivo anticancer activity

The therapeutic effectiveness of the developed formulations (5 mg/kg DOX, eq, i.v.) was assessed in breast-tumor bearing female Balb/c mice (22-25 g). Measurable tumors were found in animals after 12 weeks of DMBA dosing. Subsequently, the animals of different groups were treated with the drug and drug loaded formulations (L, LD, LDM). The first group was intravenously administered with plain DOX solution (D), while the next 3 groups were intravenously administered with a single dose of L, LD, LDM respectively. The tumor volume (V) was calculated by measuring the tumor width (w) and length (l) with a vernier caliper on an alternate day and using the following formula:
V = l×w2/ 2
The study was terminated after 28 days post-treatments. Data are shown in Figure 43.
6.4 Histopathological studies

For histopathological studies, one normal animal was sacrificed and organ tissues of the liver, kidney, Heart, and Brain were isolated. Then, a tumor-induced animal from each group was sacrificed after drug treatment (5 mg/kg DOX, eq, i.v.). The first groups received the DOX solution. While the second, third, and fourth groups were administered with L, LD, LDM respectively. After that, the organ tissues of the liver, kidney, Heart, Brain and tumor were collected. Then washed these organs and immediately fixed them in a 10% neutral buffered formalin solution for 24 hours. Tissues were cut and arranged in tissue cassettes, dehydrated, dried, and blocked by liquid paraffin. The block was cut into 3-5 µm sections, placed on a glass slide, and stained with hematoxylin and eosin (H & E) stain. Tissues were observed under a fluorescence microscope and photographed.
Moreover, the DOX-loaded surface modified liposomes (LDM) showed maximum accumulation of drug in tumor tissue due to the presence of (CR)4 cell penetrating peptide on the surface of the liposomes. This leads to a reduction in the percentage of drugs in the liver, kidney, Heart, and Brain.

The results showed that free DOX has higher access to the liver due to its higher volume of distribution, which is also reported in the literature. The PEGylation were protected from RES uptake, thus lowering the accumulation of DOX-loaded surface modified liposomes (LDM) in liver tissue. Data are shown in Figure 44.
7. Stability Studies
The aim of stability testing is to determine how the quality of a formulation varies with time under the influence of environmental conditions like temperature, humidity and light. Stability of liposomes was determined over a period of 3 months for a change in vesicle size, PDI and percent residual drug content. Samples were analyzed periodically after 0, 10, 20, 30, 45, and 90 days for vesicle size, PDI and percent residual drug content. The percent residual drug content was determined by measuring the amount of drug entrapped in the formulation after storage. Data are shown in Figure 45.
The foregoing descriptions of specific embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed, and many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described to best explain the principles of the present disclosure and its practical application, and to thereby enable others skilled in the art to best utilize the present disclosure and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the scope of the present disclosure.
, C , C , Claims:WE CLAIMS:

1. A liposome composition for targeted drug delivery to breast cancer cells, the liposome composition comprising:
a) a liposome encapsulating a therapeutic agent; and
b) (CR)4 peptide covalently conjugated to the surface of the liposome.

2. The liposome composition as claimed in claim 1, wherein the therapeutic agent is selected as doxorubicin, anti-cancer agents and a combination thereof.

3. The liposome composition as claimed in claim 1, wherein the liposomes are surface-modified with a DSPE-PEG2000-Malamide-(CR)4 conjugate.

4. The liposome composition as claimed in claim 3, wherein the DSPE-PEG2000-Malamide-(CR)4 conjugate is prepared by Mal-thiol reaction.

5. The liposome composition as claimed in claim 3, wherein the (CR)4 in DSPE-PEG2000-Malamide-(CR)4 conjugate represents a sequence of amino acids composed of (cysteine-arginine-cysteine-arginine-cysteine-arginine-cysteine-arginine), serves as a linker for the surface modification of liposomes to enable targeted delivery.

6. A method for preparing a liposome composition for targeted drug delivery to breast cancer cells, the method comprising:
a) synthesizing a DSPE-PEG2000-Malamide;
b) synthesizing a DSPE-PEG2000-Malamide-(CR)4 conjugate.;
c) preparing a surface-modified liposome (LDM) with the synthesized DSPE-PEG2000-Malamide-(CR)4 conjugate; and
d) encapsulating a therapeutic agent in the LDM.

7. The method as claimed in claim 6, wherein the therapeutic agent is selected as doxorubicin, anti-cancer agents and a combination thereof.

8. The method as claimed in claim 6, wherein synthesis of DSPE-PEG2000-Malamide involves reacting amino-PEG2000-DSPE with N-succinimide-3-(N-malamido) propionate (MPS).

9. The method as claimed in claim 6, wherein preparation of the surface-modified liposomes encapsulating anti-cancer drug by remote loading technique.