DESC:Technical Field of the Invention

[0001] Present invention generally relates to developing a novel Nano formulation of biological molecule with or without drug for drug delivery, and more particularly relates to a method of developing a novel nanoparticles formulation.

Background of the Invention

[0002] Over the past several decades the nanoparticle based drug delivery have become increasingly popular. The nanoparticle technology has enabled in the development of wide range of novel therapeutic and diagnostic platforms. The nanoparticle delivery offers many advantages such as solubilization, of hydrophobic payloads, extended blood residence times and the ability to better target a region of interest. One of the major area in which nanoparticles have particularly excelled is cancer treatment. The nanoparticle therapeutics generally relied on this passive targeting mechanism to improve efficacy over traditional free-drug formulations that often have severe systemic side effects.

[0003] Current treatment involves use of soluble or regulated biological, no combinations are available for treatment. Current oral treatment using soluble drug combination induces severe gastrointestinal and other toxicities, thus limiting continuation of therapy and patient adherence.

[0004] This Nano formulation reduces such toxicities and provides target direction of natural site of action, thus enhances efficacy. In this formulation, biological and/ or drugs are protected from non-specific effects by lactoferrin nanoparticle that further helps in localization of biological and/or drug in blood and various tissue that harbors disease and thus providing biological and/or drug affectivity against diseases.

Brief Summary of the Invention

[0005] According to a first aspect of the present invention, a novel nanoparticle formulation of biological with or without drug for targeted delivery includes: taking, required biological molecules of appropriate material. The biological molecules with or without Chemotherapeutic agents will be treated with appropriate materials derived from polymeric class for regulated release of active chemical entity will be mixed. This mixture will be added to rec. C-ter-Apo transferrin (protein)/ Apo transferrin/ C-ter-lactoferrin /lactoferrin and total volume made between 200 and 500 µL with PBS (0.1 M, pH 7.4) and then incubated on ice for 30 min.

[0006] In accordance with the first aspect of the present invention, nanoparticle preparation process further includes: a protein-biological molecule -drug mixture was slowly added to 5 to 50 ml of olive oil at 4 °C with continuous dispersion by gentle manual vortexing. The sample was sonicated at 4 °C using a narrow stepped titanium probe of ultrasonic sonicator. The sonication conditions were- 50 Amps, 3 x 5 min. pulses- 30 sec on and 30 sec off with an interval of 1 min every 5 min.

[0007] In accordance with the first aspect of the present invention, further in nanoparticle preparation process, the resulting mixture was immediately frozen in liquid nitrogen for 5 minutes and then transferred to ice and incubated for 4 h. The particles formed were pelleted by centrifugation at 8000 rpm for 15 minutes and the pellet was extensively washed with diethyl ether and dispersed in PBS and stored at 4 °C.

[0008] In accordance with the first aspect of the present invention, wherein the dispensed particles were lyophilized with and without excipient and finally the protein content in nanoparticles was estimated by Lowry method, Biological and drug molecules was estimated by chromatography.

[0009] In accordance with the first aspect of the present invention, the novel nanoparticle formulation is characterized to involve three steps, Step-1 treatment of biological molecules (plasmid DNA, SiRNA, nucleic acid, antibody, protein, peptide, oligosaccharide, etc.); Step-2 treatment of chemo-protective agent and mixing with chemotherapeutic agent; and Step-3 preparation of nanoparticles, wherein this procedure may be done with or without chemotherapeutic agent.

[0010] In accordance with the first aspect of the present invention, the novel nanoparticle formulation is characterized to use several biological molecules which are characterized by spectroscopic and electrophoretic methods. The biological molecules include but not limited to Plasmid, DNA, SiRNA, nucleic acid, antibody, protein, peptide and oligosaccharide, etc.

[0011] In accordance with the first aspect of the present invention, further the novel nanoparticle formulation is characterized to contain and use native or recombinant complete or partial portion of Apo transferrin or Lactoferrin or Transferrin nanoparticles for targeted delivery.

[0012] In accordance with the first aspect of the present invention, wherein Apo transferrin or lactoferrin or transferrin nanoparticles are loaded with protected biological molecules with or without chemotherapeutic agent and characterized in the presence of several materials include but not limited to taking lactoferrin loaded with biological molecules, Endosomal escape of lactoferrin loaded with biological molecules in presence of chemo-protective agent, polyamine oligomers etc., for site-specific localization, taking drug in combination of biological loaded lactoferrin nanoparticles, taking drug and biological molecules with loaded lactoferrin nanoparticles, regulated release of combination of drugs, drugs and biological molecules by cross linkers based preparations and taking surface modified drug, drug and biological molecules loaded lactoferrin nanoparticles with specific functional groups for specific target.
[0013] In accordance with the first aspect of the present invention, the novel nanoparticle formulation is characterized to deliver the composition with time-delayed release of biological molecules with or without chemotherapeutic agent.

[0014] In accordance with the first aspect of the present invention, wherein the prepared novel nanoparticle formulation characterization is studied through one or more ways, including Morphological characterization (FE-SEM and AFM microscopy), Loading (DL) and Encapsulation efficiency (EE), Size-exclusion chromatography (SEC), Cell viability, Gel binding assay, MTT assay, Gel Retardation assay, Cell localization assay and pH release study.
Detailed Description of the Invention

[0015] The accompanying drawings illustrate the various embodiments of compositions, methods, and other aspects of the disclosure. Any person with ordinary skills in the art will appreciate that the illustrated element boundaries in the figures represent one example of the boundaries.

FIG. 1 illustrates a flowchart depicting a novel nanoparticle formulation process according to the present invention.

FIG. 2 shows Cloning of Rec. C-ter Apo transferrin of HepG2 cell line according to the present invention.

FIG. 3 shows Expression of the rec. C-ter transferrin in E. Coli BL21 according to the present invention.

FIG. 4 shows Purification of rec. Ctd of transferrin according to the present invention.

FIG. 5 shows Size-exclusion chromatography (SEC) analysis results according to the present invention.

FIG. 6 shows FESEM analysis of Transferrin nanoparticles according to the present invention.

FIG. 7 shows FESEM analysis of rec. Ctd nanoparticles according to the present invention.

FIG. 8 shows comparison of loss and loading of drug during preparation of both nanoparticles (Apo-nanoparticles and rec. Ctd nanoparticles) according to the present invention.

FIG. 9 shows effect of pH on drug release from rec.Ctd nanoparticles according to the present invention.

FIG. 10 shows entry of the soluble drug into the cells according to the present invention.

FIG. 11 shows entry of the rec.Ctd nanoparticle drug into the cells according to the present invention.

FIG. 12 shows nanoparticle loaded with siRNA and analyzed for the encapsulation efficiency according to the present invention.

FIG. 13 shows cell viability with rec. Ctd Nanoparticles and Lipofectamine 2000 for analyzing Cytotoxicity effect of nano siRNA on cells according to the present invention.

FIG. 14 shows cell viability with rec. Ctd Nanoparticles and Lipofectamine 2000 in the presence of antibiotics according to the present invention.

FIG. 15 shows analysis of transferrin receptor (TfR) levels by conducting Cell surface receptor analysis according to the present invention.

FIG. 16 shows efficacy of siRNA loaded rec. Ctd nanoparticles according to the present invention.

FIG. 17 shows comparison of viral replication in Nano siRNA and lipofectamine mediated siRNA transfection according to the present invention.

FIG. 18 shows Plasmid isolation by alkaline lysis method according to the present invention.

FIG. 19 shows Interaction between protein and DNA at different ratios according to the present invention.

FIG. 20 shows Transmission Electron Microscopy (TEM) characterization of Blank lactoferrin nanoparticles and DNA loaded Lactoferrin nanoparticles according to the present invention.

FIG. 21 shows MTT or cell cytotoxicity assay performed in CHO cells according to the present invention.

FIG. 22 shows Gel retardation assay with different polyamines and their respective concentration according to the present invention.

FIG. 23 shows Cell localization assay performed in CHO cells according to the present invention.

FIG. 24 shows FESEM analysis of antibody loaded lactoferrin nanoparticles according to the present invention.

FIG. 25 shows Loading Efficiency Analysis of antibody loaded lactoferrin nanoparticles according to the present invention.

FIG. 26 shows pH Release Study of antibody loaded lactoferrin nanoparticles according to the present invention.

FIG. 27 shows Intracellular Antibody Delivery of AF488 labeled antibody (ab) using lactoferrin nanoparticles according to the present invention.

FIG. 28 shows MTT assay on different cell types for cytotoxicity of Ab loaded lactoferrin nanoparticles according to the present invention.

FIG. 29 shows TEM Analysis of lactoferrin nanoparticle, DNP (Daxo), CNP (Cyclo) and DCNP (Daxo+Cyclo) according to the present invention.

FIG. 30A & 30B shows pH dependent drug release of lactofeerin loaded and doxorubicin (3000A) and cyclophosphamide (3000B) nanoparticles according to the present invention.

FIG. 31 shows Cell Viability Assay of cyclophosphamide, doxorubicin and combination of (Doxo+cyclo) according to the present invention.

FIG. 32 shows cell localization Doxo and lactoferrin nanoparticles compared with free Doxo in SKNSH cells according to the present invention.

FIG. 33 shows Hydro- dynamic radius of drug (Doxo+ Curc) loaded nanoparticles according to the present invention.
FIG. 34 shows Doxorubocin+ Curcumin loaded LFNPs visualised in SK-N-SH cells and tracked by lysotracker according to the present invention.

FIG. 35 shows Hydro- dynamic radius of drug (Doxo+ Curc + Ab) loaded nanoparticles according to the present invention.

FIG. 36 shows drug and antibody (Ab) loaded nanoparticles according to the present invention.

FIG. 37 shows pH dependent drug release studies according to the present invention.

FIG. 38 shows MTT assay for Different combinational drug loaded LFNPs with and without crosslinker (MES) for cytotoxicity according to the present invention according to the present invention.

FIG. 39A and 39B shows physical characterization of surface modified lactoferrin nanoparticles by using FE-SEM and TEM analysis according to the present invention.

FIG. 40 shows Hydro Dynamic size and zeta potential of surface modified lactoferrin nanoparticles according to the present invention.

FIG. 41 shows Cell localization of surface modified nanoparticles according to the present invention.

FIG. 42 shows Enhanced Localization of Lacto-MES nanoparticles quantified by HPLC according to the present invention.

FIG. 43 shows Cell toxicity of surface modified nanoparticles according to the present invention.
Detailed Description of the Invention

[0016] The present disclosure is best understood with reference to the detailed description set forth herein. Various embodiments are discussed below. However, those skilled in the art will readily appreciate that the detailed descriptions given herein are simply for explanatory purposes as the methods and systems may extend beyond the described embodiments.

[0017] A novel nanoparticle formulation involves three steps: The method (step-1) involves in treating biological molecules (plasmid DNA, SiRNA, nucleic acid, antibody, protein, peptide, oligosaccharide, etc.) will be characterized using spectroscopic and electrophoretic methods for determination of characteristics in the presence of various materials as stabilizers, endosomal escape and site-specific localization. Our come of this step will have biological with appropriate material associated. The method (step-2) involved in treating chemotherapeutic agent with appropriated materials derived from polymeric class for regulated release of active chemical entity. The method (step-3) involved in preparation of nanoparticles, in this the products released in step-1 and with or without step 2 will be mixed.

[0018] Referring to the drawings, FIG. 1 illustrates a flowchart (100) depicting a novel nanoparticle formulation process according to the present invention. At the start process (102) takes the required biological molecules (plasmid, DNA, SiRNA, nucleic acid, antibody, protein, peptide, oligosaccharide, etc.) of appropriate material (104). Then treat the biological molecules with or without t chemo-protective agents of appropriate materials derived from polymeric class for regulated release of active chemical entity (106) and mix with the biological molecule (108). This biological mixture is further incubated with or without chemotherapeutic drug.

[0019] This mixture is then added to rec. C-ter-Apo transferrin (protein)/ Apo transferrin/ C-ter-lactoferrin /lactoferrin (110) and total volume made between 200 and 500 µL with PBS (0.1 M, pH 7.4) and then incubated on ice for 30 min (112). The protein-biological-drug mixture was slowly added to 5 to 50 ml of olive oil at 4 °C with continuous dispersion by gentle manual vortexing (114). The sample was sonicated at 4 °C using a narrow stepped titanium probe of ultrasonic sonicator (116) at sonication conditions were- 50 Amps, 3 x 5 min. pulses- 30 sec on and 30 sec off with an interval of 1 min every 5 min. The resulting mixture was immediately frozen in liquid nitrogen for 5 min (118) and then transferred to ice and incubated for 4 h (120). The particles formed were pelleted by centrifugation at 8000 rpm for 15 minutes and the pellet was extensively washed with diethyl ether and dispersed in PBS and stored at 4 °C (122). Then the dispensed particles lyophilized with and without excipient (124). At the end (128), the protein content in nanoparticles was estimated by the Lowry method, Biological and drug was estimated by chromatography (126).

[0020] The prepared nanoparticle formulation is characterized to use native or recombinant complete or partial portion of Apo transferrin or Lactoferrin or Transferrin nanoparticles that are loaded with protected biological with or without chemotherapeutic agent and characterized in the presence of several materials such as taking lactoferrin loaded with biological molecules and endosomal escape of lactoferrin loaded with biological molecules in presence of chemo-protective agent, polyamine oligomers etc., for site-specific localization. Various studies and analysis are conducted by using one or more ways including: Morphological characterization (FE-SEM and AFM microscopy); Loading (DL) and Encapsulation efficiency (EE); Size-exclusion chromatography (SEC); Cell viability; Gel binding assay; MTT assay; Gel Retardation assay; Cell localization assay and pH release study.

[0021] Studies: Morphological characterization (FE-SEM and AFM microscopy)
Structure, size and morphology of NP were characterized using various sources such as FESEM (Field Emission Scanning Electron Microscope), AFM (Atomic Force Microscopy). For FE-SEM the freshly prepared NP (NP suspension) were dried on a sterile glass slide; particles were coated with gold. For AFM imaging the NP was spin coated on a clean glass slide. For 3 all the imaging technique, instruction were followed according to manufacturer for the data collection and their analysis.

[0022] In vitro studies: Loading (DL) and Encapsulation efficiency (EE)
EE was defined as the ratio of actual and initial amount of drugs encapsulated in NP. Drugs entrapped in Lf-NP was quantified using HPLC. Freshly prepared NP was incubated with 1ml of PBS (pH5) at room temperature in rocking condition for 24hr. After drugs were getting released into the solution, 30% of silver nitrate (100µl) per ml of sample was added to precipitate the protein content. Further drug was extracted by adding 1ml of HPLC grade methanol, mixture was centrifuged at 12000rpm for 20min and drugs were quantified in the supernatant. Experiment was done in a set of three. A standard curve was established using different known concentration of AZT, EFV and 3TC estimated using HPLC. Encapsulation was calculated using below mentioned formula. Biological will be assessed using appropriate method. Encapsulation Efficiency (EE) = (Existent amount of drugs in NP/ Initial amount of drug used in NP preparation) x 100%.

[0023] Example.1: Formulation containing siRNA loaded recombinant apotranferrin nanoparticles.
FIG. 2 shows Cloning of Rec. C-ter-Apo transferrin of HepG2 cell line (200) according to the present invention. In this total RNA was isolated from the HepG2 cell line, cDNA was synthesized, and C-ter domain was amplified as mentioned in the materials and methods. Results show successful PCR amplification of the C-ter region of the transferrin ORF (Fig. A). This amplicon was sub cloned into a pDual GC vector (Fig. B) which can over express the cloned DNA fragment in both mammalian and E.Coli expression systems (dual expression vector) between two restriction sites of Eam1104I. Resulting recombinant plasmid which was confirmed by PCR was further confirmed by restriction enzymatic digestion with Eam1104I (Fig. C) showing successful release of the cloned DNA fragment from the recombinant plasmid.

[0024] FIG. 3 shows Expression of the rec. C-ter transferrin in E. Coli BL21 (300) according to the present invention. The spDual GC harboring C-ter region of the human transferrin was transformed to E.Coli BL21 (DE3-Lac-I) and PCR positive colonies were selected. Selected single colony was cultured and induced for the expression of rec.protein with IPTG of mentioned concentrations. Samples were collected at mentioned time points lysed and analyzed. Results show over expression of the rec. protein in Coomassie stained SDS-PAGE in the position ~45 kDa with respect to the 0 h induction control sample (Fig. A). The expression of the ~45 k Da rec. protein was further confirmed in western blot (Fig. B) based on the presence of the poly-Histidine in the rec.protein by using poly-Histidine antibody.

[0025] FIG. 4 shows Purification of rec. Ctd of Apo transferrin (400) according to the present invention. The proteins in the crude E.Coli cell lysate was precipitated with mentioned percentages of ammonium sulphate ((NH4)2SO4) and a small fractions of the resulting protein pellets were boiled in 1xSDS loading buffer and analyzed in 10% SDS-PAGE (Fig. A) showing the precipitation of the ~45 kDa protein in 20% cut off/pellet along with other proteins which was absent in remaining pellets and the final resulting supernatant. Western blot analysis of the 20% cutoff pellet (Fig. B) showing the ~45 kDaprotein was the rec.protein. These results show the precipitation of the major part of the rec.protein with 20% ((NH4)2SO4) concentration.

[0026] FIG. 5 shows Size-exclusion chromatography (SEC) analysis (500) according to the present invention. The rec.Ctd of Tf from the 20% ammonium sulphate pellet was further purified to eliminate the co-precipitated proteins. The resulting pellet was subjected to Size-exclusion chromatography (SEC) as described in methods. The resulting protein samples were boiled in SDS loading buffer and analyzed in 10% SDS-PAGE by silver staining. Results showed the elutes from 41 to 48 are expressing ~45 kDa rec.protein without any other contaminant proteins. These eight elutes were pooled, concentrated and stored at -70 °C for further use.

[0027] FIG. 6 shows FESEM analysis of Transferrin nanoparticles (600) according to the present invention. Nanoparticles prepared with commercially obtained human Transferrin protein were spread and dried on 0.5 x 1.0 cm microscopic glass slide piece, processed and subjected to FESEM. The resulting data shows the size and shape of the nanoparticles loaded with SiRNA. Even though different size populations were observed, all are in nearly spherical shape and the average size of the nanoparticles was ~70 nanometers (nm).

[0028] FIG. 7 shows FESEM analysis of rec. Ctd nanoparticles (700) according to the present invention. Nanoparticles prepared with purified rec. C-ter protein were spread and dried on 0.5x1.0 cm microscopic glass slide piece, processed and subjected to FESEM. The resulting data shows the size and shape of the nanoparticles loaded with SiRNA. Even though different size populations were observed, all are in nearly spherical shape and the average size of the nanoparticles was ~25 nm.

[0029] FIG. 8 shows comparison of loss and loading of drug during preparation of both nanoparticles (Apo-nanoparticles and rec. Ctd nanoparticles) (800) according to the present invention. We measured the amount of drug encapsulated in both nanoparticles (Tf nanoparticles and rec.Ctd nanoparticles) and also measured drug retention in the oil and aqueous phases in nanoparticles preparation by taking equal amounts by measuring the O.D at 490 nm (property of drug) and the mean±SD were plotted against each other. Analysis shows the drug loading capacity varies and is nearly 30% more drug was encapsulated in rec.Ctd nanoparticles. Analysis revealed that even though the loss of drug in oil and aqueous phase is same from both preparations, majority drug loss was observed during the PBS wash of the nanoparticles.
[0030] FIG. 9 shows effect of pH on drug release from rec.Ctd nanoparticles (900) according to the present invention. The effect of pH on drug release was measured at different time points. For this, Rec.Ctd nanoparticles were incubated in PBS of pH 2.0, 5.5, 7.4 and 9.0 for 0 to 6 h. in triplicates and the amount of drug released from each sample was measured. The amount of doxorubicin released by nanoparticles were estimated by spectrophotometer, optical density read at 490 nM wave length and the results were plotted as mean±SD against each other. Results showed that nanoparticles at pH 2.0 released maximum amount of drug in the 1st h of post incubation. In case of nanoparticles at pH 5.5 the drug release was slow, study and continuous for longer periods. But, at alkaline pH drug release from nanoparticles was absent.

[0031] FIG. 10 shows entry of the soluble drug into the cells (1000) according to the present invention. For this, the HeLa cells were grown on the microscopic cover slips and incubated with drug. After 1 h of post incubation, cells were processed and co focal microscopic images were taken and analyzed. The drug (doxorubicin: red fluorescence) spread evenly in the cytoplasm and nucleus however the amount of accumulation of drug was more at nuclear membrane. It shows that drug directly diffused through the cell. DAPI (Blue) used as nuclear counter stain. Merged images of all the fluorescence are shown. All the scale bars (5000 nm) are indicated as inlets. Results showed, at 1 h of post incubation, the drug spread evenly in the cytoplasm and nucleus; however drug was more concentrated at the nuclear membrane. It shows that drug directly diffused through the cell.

[0032] FIG. 11 shows entry of the rec.Ctd nanoparticle drug into the cells (1100) according to the present invention. For this, the HeLa cells were grown on the microscopic cover slips and were incubated with nanoparticles prepared with rec.Ctd protein. After the mentioned time of post incubation, cells were processed and confocal microscopic images were taken and analyzed,

[0033] Photo micrograms taken at 1h of post incubation, nanoparticles were accumulated on the cell wall and very little amount of drug (doxorubicin: red fluorescence) entered into cells. But at 2 h of post incubation, more amount of drug accumulated in the cytoplasm when compared to nucleus however accumulation of drug is observed on the nuclear membrane. Immunofluorescence analysis of HIV-1 RT, Topo IIa and Topo IIß (first to third vertical lanes) at 0, 1, 2 and 4 h after infection with HIV-1. HIV-1 RT tagged with AF 594 secondary antibody (Magenta). Topo IIa and Topo IIß were directly tagged with TRITC (Red) and FITC (Green) respectively. DAPI (Blue) used as nuclear counter stain (fifth vertical lane). Merged images of all the fluorescence shown in fourth panel. All the scale bars (10 µm) are indicated as inserts. DAPI (Blue) used as nuclear counter stain. Merged images of all the fluorescence are shown. All the scale bars (5000 nm) are indicated as inlets.

[0034] Results shows that, at 1 h of post incubation, nanoparticles were accumulated on the cell wall and very little amount of drug entered into cells. But at 2 h of post incubation, higher amount of drug spread in the cytoplasm and fewer amounts were observed in the nucleus and also the accumulation of drug on the nuclear membrane was observed. It shows that controlled release of the drug from the nanoparticles.

[0035] FIG. 12 shows nanoparticle loaded with siRNA and analyzed for the encapsulation efficiency (1200) according to the present invention. Nanoparticles loaded with siRNA were analyzed for the encapsulation efficiency. In (Fig. A) Silver stained SDS-PAGE gel showing siRNA extracted from nano formulation using TRI reagent when compared to control siRNA the arrow indicates the position of siRNA in the gel. Densitometric analysis of the bands in silver stained gel showing percent availability of siRNA is shown in (Fig. 1B). Results show that, in comparison to the input siRNA used in the preparation of nanoparticles, the siRNA present in the nanoparticles was ~ 80%. The remaining 20 % may be the loss during the process of nanoparticles preparation.

[0036] FIG. 13 shows cell viability with rec. Ctd Nanoparticles and Lipofectamine 2000 for analyzing Cytotoxicity effect of nanosiRNA on cells (1300) according to the present invention. Cells were treated with Nanoparticles and Lipofectamine 2000 in a sufficient quantity to transfect 200 nM of siRNA. After 16 h of incubation, cell viability was estimated using standard MTT assay and results were plotted. Cytotoxicity effect of nanosiRNA on cells. Percent viability in cells transfected with nanosiRNA when compared with lipofectamine mediated delivery of siRNA in cells. Untransfected cells used as negative control. Values are mean ± SEM, n=3. The statistical significance is denoted by * at p value = 0.05. Results from this data showed that, >97% of the nanoparticle treated cells were alive in comparison to control cells. But in case of lipofectamine 2000 treatment, cell viability was 80% only.

[0037] FIG. 14 shows cell viability with rec. Ctd Nanoparticles and Lipofectamine 2000 in the presence of antibiotics (1400) according to the present invention. Cells were treated with Nanoparticles and Lipofectamine 2000 in a sufficient quantity to transfect 200 nM of siRNA in the presence of antibiotics. After 16 h of incubation, cell viability was estimated using standard MTT assay and results were plotted. Effect of antibiotics in nano and lipofectamine mediated siRNA delivery. Percent viability in cells transfected with nanosiRNA when compared with lipofectamine mediated delivery of siRNA in the presence of antibiotics. Untransfected cells used as negative control. Values are mean ± SEM, n=3. The statistical significance is denoted by * at p value = 0.05. Results from this data shows that, >85% of the nanoparticle treated cells were alive in comparison to control cells which were also in antibiotic stress. But in case of lipofectamine 2000 treatment, cell viability was 50% only.

[0038] FIG. 15 shows analysis of transferrin receptor (TfR) levels by conducting Cell surface receptor analysis (1500) according to the present invention. PBMCs were infected with HIV-1 and samples were collected at different time points to analyze cell surface receptor with flow cytometer. PBMCs infected with HIV-1 were collected at mentioned time points labeled with TfR antibody. Data plotted as counts on Y-axis and fluorescence intensity on X-axis. Results indicated that, up to 4 h of post infection, there was no significant change in the TfR levels however a non-significant change was observed from 12 h onward. At 24 h of post infection, a significant decrease in the cell surface TfR levels was evident.

[0039] FIG. 16 shows efficacy of siRNA loaded rec. Ctd nanoparticles (1600) according to the present invention. Cells were transfected with siRNA specific to Topo IIß using nanoparticles and Lipofectamine 2000. Samples were collected at different time points and analyzed for the knockdown efficiency by western blot. Topo IIß Protein levels in nano formulated siRNA treated cells were decreased considerably by 24 h of post transfection and the levels were further decreased by 48 h and reached minimum. At 72 h of post treatment the protein levels were slightly increased in comparison to 48 h (Fig. A). Topo IIß protein levels at different time points in siRNA delivery using Lipofectamine 2000 (Fig. B). ß-actin levels are shown as loading control, even though the protein levels decreased at 24 h and further at 48 h but not as effective as nano formulation. Later at 72 h of post treatment, protein levels were re-assuming quickly in comparison to nano formulation.

[0040] FIG. 17 shows comparison of viral replication in nano siRNA and lipofectamine mediated siRNA transfection (1700) according to the present invention. HIV-1 replication was analyzed in Topo IIß down regulated cells using nano and lipofectamine mediated siRNA delivery. After 96 h post infection, virus replication was studied using p24 assay. Viral replication measured by p24 assay in cells infected with HIV-1 after Topo IIß down regulation using nano and lipofectamine mediated siRNA delivery. Viral replication monitored by p24 ELISA at 4th day post infection. GFP-siRNA used as siRNA negative control, uninfected/untransfected cells used as experimental negative control and HIV-1 infected cells used as positive control. Lipofectamine 2000 used as delivery control. Un-infected cell supernatant used as negative control for ELISA. Values are mean ± SEM, n=3. The statistical significance is denoted by * at p value = 0.05.

[0041] These results demonstrate that nanoparticles prepared with Ctd of transferrin are small, holding more amount of siRNA and executed the receptor mediated delivery as like native nanoparticles. These Ctd nanoparticles are capable of delivering the siRNA to the target cells through the receptor mediated delivery and have potential for site directed delivery system. Results indicated that, in lipofectamine mediated siRNA delivery the viral replication was 27%, whereas with nanosiRNA delivery the viral replication was significantly decreased to 5.8%. Hence, nanosiRNA could successfully decrease the viral replication and hence nano formulation can be used for efficient siRNA delivery.

[0042] Example 2. Formulation containing DNA loaded lactoferrin nanoparticles. FIG. 18 shows Plasmid isolation by alkaline lysis method (1800) according to the present invention. The plasmid has been successfully isolated by alkaline lysis method. The Gel picture showed 4.7 Kb pEGFP-C1 plasmid.

[0043] Gel Binding Assay: To optimize the ratio of protein and DNA for the preparation of nanoparticles, the experiment was performed. Increasing ratios of protein and DNA was used as per the Table No 1. The assay was performed by keeping the concentration of DNA constant and varying the concentration of lactoferrin protein.

DNA Protein
1 (6.9 µg) 1 (6.9 µg)
1 (6.9 µg) 2 (13.8 µg)
1 (6.9 µg) 4 (27.6 µg)
1 (6.9 µg) 6 (41.4 µg)
1 (6.9 µg) 8 (55.2 µg)
1 (6.9 µg) 10 (69 µg)

Table 1. Ratio of DNA and protein for gel binding assay.

[0044] FIG. 19 shows Interaction between protein and DNA at different ratios (1900) according to the present invention. The DNA and protein were incubated for one hour and analyzed for appropriate concentration of protein for entrapment of DNA by gel electrophoresis. Interaction between protein and DNA at different ratios (M- marker, 1-10 – ratio of various concentration of protein according to table no.1, C- control i.e. only plasmid. Results show the interaction between different concentrations of lactoferrin with a constant concentration of DNA. Gel picture shows at concentration of 1:8 and 1:10 ratio the DNA band is not visible suggest the entrapment of DNA into the protein.

[0045] Nanoparticle Preparation: DNA loaded lactoferrin nanoparticles were prepared through sol-oil chemistry. 1:10 ratio of DNA and protein was taken in a 5 ml tube and incubate for 1 hr. Then mixed with 4 ml olive oil in the above mixture, while vortexing. The mixture was sonicated for 15 min at 60 amp. Immediately exposed to liq. Nitrogen for 10 minutes. Thawed the above sample in ice for 4hrs. Centrifuge for 10 min in 4 °C at 6000 rpm. Wash the pellet with Diethyl ether. Resuspended in 1x TE buffer. The Nanoparticle morphology were examined using transmission electron microscope (TEM) operated at 80 KV. TEM sample was prepared by fixing the sample on 200 mesh type-B carbon coated copper grid using 2% osmium tetroxide in 50 mM phosphate buffer followed by staining with phosphotungstic acid. The characterization and data analysis was done according to the protocol described as per the instructions.

[0046] FIG. 20 shows Transmission Electron Microscopy (TEM) characterization of Blank lactoferrin nanoparticles and DNA loaded Lactoferrin nanoparticles (2000) according to the present invention. Lactoferrin nanoparticle and DNA loaded lactoferrin nanoparticle were analyzed by transmission electron microscopy. The size has been increased after loading of DNA into nanoparticle.

[0047] FIG. 21 shows MTT or cell cytotoxicity assay performed in CHO cells (2100) according to the present invention. MTT or cell cytotoxicity assay was performed in CHO cells. Approx. ten thousand cells were seeded in the 96 well plate. The cells were treated with increasing concentration (50, 100, 250, 500, 750 and 1000ng) of DNA loaded LFNP. The cells were incubated for 24h in CO2 incubator. Then the media were replaced by 200µl of MTT (5mg/ml) per well and incubated for 4h. After 4h the formazon crystal formed was dissolved in 200 µl of DMSO. The absorbance was recorded using ELSA reader at 570nm. Result shows the nontoxic behavior of lactoferrin nanoparticle. Because even at higher concentration (1000 ng) the cells are viable.

[0048] FIG. 22 shows Gel retardation assay with different polyamines and their respective concentration (2200) according to the present invention. Gel retardation assay is performed to check the binding of different polyamines (DNA protecting agents) with DNA. For the assay following procedure has followed: 0.75µg of DNA has been incubated with different polyamines with different concentrations like Spermidine (300ng and 1000ng), Tetraamine (300ng and 1000ng) and Hexamine (300ng and 1000ng) separately, for 1hr at 37°C in shaking condition. After incubation gel electrophoresis, has performed in order to check the binding of the polyamines with DNA.

LANE-1:- Ladder.
LANE-2:- Undigested plasmid.
LANE-3:- Digested plasmid.
LANE-4:- Spermidine 300ng concentration.
LANE-5:- Spermidine1000ng concentration.
LANE-6:- Tetramine 300ng concentration.
LANE-7:- Tetramine 1000ng concentration.
LANE-8:- Hexamine 300ng concentration.

[0049] FIG. 23 shows Cell localization assay performed in CHO cells (2300) according to the present invention. To localize the nanoparticles intracellularly, this assay has been performed. The assay was done in CHO cell. Briefly, the 0.01 million cells were seeded on the cover slip and incubated for 12h to allow the cells to adhere to the cover slips. After 12h, the cells were treated with 1) Only GFP DNA mixed with transfecting agent (Lipofectamine 2000c) and 2) GFP DNA loaded LFNP. Blank LF-NP was used a positive control. The cells were incubated for 48h followed by fluorescent analysis using fluorescence microscope.

[0050] The assay was performed using fluorescence microscopy. Result shows more localization of DNA loaded lactoferrin nanoparticle as compare to naked DNA in combination with transfecting agent. DNA loaded Lacto-NP showed better GFP expression (green) compared to lipofectamine mediated DNA delivery. Cells treated with Hexamine-DNA polyplex Lacto-NP showed higher GFP expression (green) compared to Tetramine-DNA polyplex Lacto-NP. This suggests increase in size of polyamine oligomer increases efficiency of DNA delivery.

[0051] Example 3. Formulation containing antibody loaded lactoferrin nanoparticle.
Preparation of Antibody loaded Nanoparticles: A sample containing 7 µl of 50µg/µl lactoferrin was mixed with 1 µl of 2µg/µl Alexa-Fluor 488 goat anti-mouse IgG and volume made upto 250 µl by PBS (pH=7.4) and incubated for at least 15 minutes. Then 1 ml of olive oil was added to it and it was mixed well. It was sonicated at 50 ampere; 30 pulse on ice with 30 seconds on and off cycle for 5 minutes, repeated three times, with an interval of 1 minute each time. Then sample exposed to liquid nitrogen for 10 min for particles to precipitate. Particles were kept at room temperature, then pelleted at 8000 rpr for 20 minutes. The upper oil layer was removed. The lower aqueous layer was removed carefully by using a 1 ml tip attached to 10 µl tip, not to disturb the nanoparticle pellet. The aqueous layer was stored separately for loading efficiency analysis. The pellet thoroughly washed with diethyl ether vigorously thrice to remove the left over oil present. It was allowed to dry so that all diethyl ether get evaporated. Then, 1 ml of PBS (pH=7.4) was added to it and it was sonicated for around 30 seconds. Three different samples were prepared with 1µl, 5µl and 10µl of antibody to analyze the loading efficiency of the nanoparticle. For this the FESM analysis is performed which is explained below.

[0052] FIG. 24 shows FESEM analysis of antibody loaded lactoferrin nanoparticles (2400) according to the present invention. The nanoparticle was filtered with 0.2 µm filter and spread on small glass slide piece cut out of a glass slide using a glass slide cutter. The glass piece was attached to a double sided tape fixed to a petridish and was taken for SEM analysis. The Photo microgram showed the size and shape of the antibody loaded lactoferrin nanoparticles. Average size of the nanoparticles is ~36 nm. The range is 20nm-42nm.

[0053] FIG. 25 shows Loading Efficiency Analysis of antibody loaded lactoferrin nanoparticles (2500) according to the present invention. We have taken aqueous phase of each nanoparticle preparation and made up to 1000 uL of PBS-pH7.4 and fluorescence reading was measured. PBS-pH7.4 used as blank. Considering dilution factor the values given are the % of loading efficiency. Antibody used in the preparation is 0-10uL of AF- 488 (1uL=2ug). 1ul of AF488 gives: 920,000 FU. 350ug of the lactoferrin used in each preparation. Several concentrations such as 1µl/ml , 0.5µl/ml,0.25 µl/ml,0.125 µl/ml and 0.0625 µl/ml was prepared by adding 1 µl of Alexa-Fluor 488 goat anti-mouse IgG and serially diluting it. The respective fluorimeter readings were taken with excitation wavelength of 490 nm and emission wavelength of 535 nm.

Concentration Fluorimeter Reading
1µl/ml 1200 (over-shoots)
0.5µl/ml 467.64
0.25 µl/ml 230.08
0.125 µl/ml 115.39
0.0625 µl/ml 58.04

Above results showed that 1µl (1µg) of AF488 gives 920,000 FU.

[0054] FIG. 26 shows pH Release Study of antibody loaded lactoferrin nanoparticles (2600) according to the present invention. The 100 µl of 10 µl antibody loaded nanoparticle was added to 1000 µl of PBS (pH=5.5) and fluorimeter readings were taken at 0 minute, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours and 4 hours with excitation wavelength of 490 nm and emission wavelength of 535 nm. We have taken 100uL (1/10) of nanoparticle preparation and added to 1000 uL of PBS-pH5.5 and incubated for the mentioned time. At each time point, we measured the fluorescence and plotted. PBS-pH5.5 used as blank. Considered dilution factor, the values given are the 1/1000 of obtained. Antibody used in the preparation is 10uL of AF- 488 (20 ug). 1ul of AF488 gives: 920,000 FU.

[0055] FIG. 27 shows Intracellular Antibody Delivery of AF488 labeled antibody (ab) using lactoferrin nanoparticles (2700) according to the present invention. The SupT1 cells were maintained in RPMI-1640 complete media by passaging as per requirement. When 70 % confluence is achieved, the 0.5 million cells were seeded to 12 well-plate, for treatment. 100 microlitre of the nanoparticle prepared was added to it and it was incubated for 14 hours duration. Then the cells were pelleted and given PBS washes. Then a drop of the cell suspension is put on the glass slide and cover slip was added to it and visualized under confocal microscope at 488 nm. (forAlexa-Fluor 488 goat anti-mouse IgG). SupT1 cells were treated with the nanoparticles loaded with AF488 labeled ab in the cell culture medium and after 14hrs, images were acquired using confocal microscope.

[0056] FIG. 28 shows MTT assay on different cell types for cytotoxicity of Ab loaded lactoferrin nanoparticles (2800) according to the present invention. The MTT assay of different cell types for cytotoxicity of Ab loaded LFNPs are measured at 15hr time point. Comparing the cytotoxicity in different cell groups; Adherent(SK-N-SH), Suspension (Y79) and mixed (Colo), we have found that Ab inhibiting Topoisomerase alpha (cell proloferation) has different way of affecting when delivered via Lactoferrin nanoparticles. The susceptibility are increasing in the order of Y79>Colo>SK-N-SH when compared at the standardized time point of 15th hour.

[0057] The prepared nanoparticle formulation is characterized to use native or recombinant complete or partial portion of apotransferrin or lactoferrin or transferrin nanoparticles that are loaded with protected biological with or without chemotherapeutic agent and characterized in the presence of several materials such as taking drug in combination of biological loaded lactoferrin nanoparticles, taking drug and biological molecules with loaded lactoferrin nanaoparticles and regulated release of combination of drugs, drugs and biological molecules by cross linkers based preparations.

[0058] Example 4: Lactoferrin loaded doxorubicin, cyclophosphamide nanoparticles:
Isolation of Lactoferrin from Milk, Bovine milk was centrifuged at 10,000 rpm for 10 min @ 4 degree Celsius. HCl was added to supernatant to get precipitate. After getting precipitate it was again centrifuged at 10,000 rpm for 10 min @ 4 degree Celsius. pH of supernatant was adjusted to 4.6. Supernatant was put into dialysis membrane (50kDa cut off) and kept in sucrose bed overnight. The supernatant was taken from dialysis membrane and run on SDS PAGE to check our protein of interest.

[0059] Preparation and characterization of Lactoferrin loaded doxorubicin and cyclophophoamide nanoparticles: Preparation of combination cancer nanoparticles: Doxorubicin (Doxo) and Cyclosphophamiade (Cep) incubated with lactoferrin to the saturation kinetics, then added to olive oil, while stirring. Contents were sonicated frozen quickly to obtain nanoparticles. Nanopartciles were thoroughly washed with diethyl ether, dried and used for experiments.

[0060] FIG. 29 shows TEM Analysis of lactoferrin nanoparticle, DNP (Daxo), CNP (Cyclo) and DCNP (Daxo+Cyclo) (2900) according to the present invention. The structure of nanoparticle is analysed by TEM analysis. In this Lf NPs, DNP (Daxo), CNP (Cyclo) and DCNP (Daxo+Cyclo) were analyzed by using the Transmission Electron Microscope. The range of diameter for Lactoferrin nanoparticle was found to be 25-30nm and for drug loaded lactoferrin nanoparticle was found to be 45-90nm.
[0061] Encapsulation Efficiency: From the freshly prepared NPs 100ul was taken and it was incubated in 1ml of PBS for 30min. At the end of 30 min, 100ul of 30% silver nitrate was added to precipitate the protein. To extract the drug, 1ml of water was added and the mixture was centrifuged @12000 rpm for 15min.At the end of centrifugation, the supernatant was collected and filtered using 0.2 micron syringe filters and the filtrate obtained was estimated using HPLC. Encapsulation efficiency was calculated using the formula:
Encapsulation efficiency: (Mtotal-Mlost/ Mtotal) x 100
Encapsulation efficiency of Lactodoxonano was found to be 60-65%
Encapsulation efficiency of Lactocypnano was found to be 70-75%.

[0062] The NPs pellet was suspended in 1ml 1X PBS (in the range 1-9). It was kept for incubation on a rocker at room temperature for 2 hours. After that 300ul of 30% silver nitrate was added and the drug was extracted by adding 1ml of 1X PBS. The above mixture was centrifuged at 12000 rpm for 15min @ 4 degree Celsius. The supernatant obtained was filtered using 0.2 micron syringe and the filtrate was estimated by HPLC.

[0063] FIG. 30A & 30B shows pH dependent drug release of lactofeerin loaded and doxorubicin (3000A) and cyclophophoamide (3000B) nanoparticles according to the present invention. The pH dependent drug release assay for Lactodoxonano was incubated in buffers of different pH. The release of doxorubicin was maximum at pH 5 as shown in (Fig. 30A). The pH dependent drug release assay for Lactocyclonano was incubated in buffers of different pH. The release of cyclophosphamide was maximum at pH 5 as shown in (Fig. 30B).

[0064] FIG. 31 shows Cell Viability Assay of cyclophophoamide, doxorubicin and combination of (Doxo+cyclo) according to the present invention. SKNSH cells (0.2X105/ well) were seeded in a 96 well plate and were incubated at 37o C for 4 hrs in a CO2 incubator. These cells were incubated with increasing concentrations of both Cyclophosphamide and Doxorubicin in soluble forms and incubated for 16 hrs. After discarding the media the cells were resuspended in a new medium. To this, 20ul of 5mg/ml MTT was added and was incubated for 4hrs.After the medium was removed the cells was dissolved in DMSO and was read in an ELISA microplate reader at 570 nm.

[0065] In this K-562 cells is treated with different concentrations of soluble Doxo, sol Cyclo, sol (Doxo+cyclo) and equivalent dosage of the drug in DLfNPs, CLfNPs and DCLfNPs. It showed a reduced IC50 for drug-LfNPs as compared to free drug for K-562 cell line. Compared to soluble drugs, nano formulated drugs are more effective in lower concentrations. The CC50 of sol doxo is 22.5055 µM, sol cyclo is 30.8997 µM and sol.doxo+cyclo 23.2963 µM , but in case of nano formulation drugs nano doxo was seen to be CC50 7.873 µM, nano cyclo was observed to be 6.3693 µM and nano daxo+cyclo was seen to be 7.3177 µM.

[0066] FIG. 32 shows cell localization Doxo and lactoferrin nanoparticles compared with free Doxo in SKNSH cells (3200) according to the present invention. The time based cellular uptake of Doxo and lactoferrin nanoparticle is compared with free Doxo in SKNSH cells. Time course experiment showed that nanoparticles are retained for longer time (till 8 hr) in cells as compared to free Doxo that gets cleared after 8 hr. Compared to sol drug, higher localization of nano formulated drug was observed at all-time points. Maximum availability of sol drug was observed at 2h, but in case of nano drug maximum was observed to be at 4h and the presence of drug was seen even at 8Hr time point. The drug releasing from nanoparticles is slow compare to sol. form so drug is available long time effectively.

[0067] FIG. 33 shows Hydro- dynamic radius of drug (Doxo+ Curc) loaded nanoparticles (3300) according to the present invention. Hydro-dynamic radius of doxo and curcumin loaded nanoparticles size is 132nm in PBS.

[0068] FIG. 34 shows Doxorubocin+ Curcumin loaded LFNPs visualised in SK-N-SH cells and tracked by lysotracker (3400) according to the present invention. The drugs Doxorubocin+ Curcumin loaded lactoferrin nanoparticles are visualised in SK-N-SH cells with (upper panel) and without (lower panel) in lysotracker under fluroscence microscope at 63x magnification. The drug loaded nanoparticles enter into the cells through endosomal mechanism that was tracked by lysotracker.

[0069] FIG. 35 shows Hydro- dynamic radius of drug (Doxo+ Curc + Ab) loaded nanoparticles (3500) according to the present invention. Hydro-dynamic radius of Drug (Doxo+ Curc) +Ab loaded nanoparticles size is 111nm in PBS.

[0070] FIG. 36 shows drug and antibody (Ab) loaded nanoparticles (3600) according to the present invention. The Secondary Ab (tagged with Alexa fluor-594) is delivered in combination with doxorubicin and Curcumin using lactoferrin nanoparticle and counter stained with DAPI (blue) and Lysotracker (red) at 63x magnification. The drug and antibody (Ab) loaded nanoparticles enter into the cells through endosomal mechanism that was tracked by lysotracker.

[0071] Cross Linked Lactoferrin Np for regulated drug release:
FIG. 37 shows pH dependent drug release studies (3700) according to the present invention. The pH Dependent release studies were measured for Physiological Conditions. The pH dependent release of cross linked Doxo loaded Lacto-NP was studied at pH 5.5 and Ph 7.4 at 4hr time point. Drug loaded Nanoparticles with MES is incubated for 20 minutes and pH dependent release profile is measured at 4hr time point. The release of Doxo in Lacto-MES NP at 4hr was slower than Lacto NP. The drug release from nanoparticles was maximum at pH5.5, but crosslinker (MES) treated nanoparticles drug release was seen at pH 5.5 less compare to without crosslinker. The crosslinker treated nanoparticles release drug slowly with long period thus increasing the drug availability.

[0072] FIG. 38 shows MTT assay for Different combinational drug loaded LFNPs with and without MES for cytotoxicity (3800) according to the present invention. The MTT assay with Different combinational drug loaded LFNPs with and without MES for cytotoxicity. All the Nanoparticles and their crosslinked counterparts are compared and MES treated ones are less cytotoxic with 16 hours incubation in cells. This suggests slow release of drugs in crosslinked Lacto-NP in comparison to Lacto-NP. The crosslinker (MES) treated drug loaded nanoparticles shows less toxicity at any concentration compare to, without crosslinker (MES) .which shows slow drug in crosslinker treated nanoparticles .
[0073] Example 5: Preparation of surface modified lactoferrin nanoparticles with sodium -2-mercaptoethane sulphonate (MES): Drug loaded lactoferrin nanoparticles were prepared through sol-oil chemistry. 2.5milligram of drug was dissolved in 1000µl of Milli-Q water and was gently mixed with 10 mg of lactoferrin dissolved in 1ml of ice cold phosphate buffer saline (pH 7.4). Mixture was incubated in ice for an hr and then added sodium -2-mercaptoethane sulphonate (MES) in different ratio (up to 2×104MES: lactoferrin mole excess). Then it was slowly added to 25ml of olive oil with gentle vortexing. The sample was sonicated for 15 misn at 4°C with the help of narrow stepped titanium probe of ultrasonic homogenizer (300V/T, Biologics Inc., Manassas, Virginia, USA). Resulting mixture was immediately transferred in liquid nitrogen for 10 min then thawed on ice for 4hr. Particles formed were centrifuged at 6000 rpm for 10 min at 4°C. Pellet formed was extensively washed twice with ice cold diethyl ether (to completely remove oil) and then dispersed in 1ml of PBS and further removing excess of MES by washing with PBS.

[0074] FIG. 39A and 39B shows physical characterization of surface modified lactoferrin nanoparticles by using FE-SEM (3900A) and TEM (3900B) analysis according to the present invention. The shape and morphology of the particles were characterized by field emission scanning electron microscopy (FE-SEM, operated at 5 KV) and TEM. For the SEM analyses a sample (10 µl) of the particles was spotted onto a glass wafer, followed by drying and gold sputtering. For the TEM analyses a sample of the particles was spotted onto a 200 mesh type-B of carbon coated copper grid and then stained with uranyl acetate(30s) followed by drying. Results suggest that the size of surface modified Blank Np is ~40nm and surface modified Drug Loaded Np is ~90nm.

[0075] FIG. 40 shows Hydro Dynamic size and zeta potential of surface modified lactoferrin nanoparticles (4000) according to the present invention. The size and the size distribution of the particles were determined by Malvern Nano sight in PBS buffer. The charge of the molecules was calculated by zeta sizer. Results showed that MES nanoparticle and ~120 nm for drug loaded Lacto-MES NP. The charge was observed to be -3 mV for lacto-NP and -12mV for Lacto-MES NP.

[0076] FIG. 41 shows Cell localization of surface modified nanoparticles (4100) according to the present invention. HL2/3 cells were seeded and allowed to adhere. Cells were incubated with Drug loaded nanoparticles loaded with 4µg of Doxorubicin at increasing time points. The cells were washed 3 times with 1xPBS. The images were captured with Carl-Zeiss Confocal microscope. The results suggest a slow increase in the drug localization of Doxorubicin from 1hr time point, releasing maximum at 4hr, in case of Lacto-MES NP. Lacto NP showed highest and sudden release of drug at 4hr point.

[0077] FIG. 42 shows Enhanced Localization of Lacto-MES nanoparticles quantified by HPLC (4200) according to the present invention. The 2* 106 HL2/3 cells were seeded and allowed to adhere. Cells were incubated with Drug loaded nanoparticles loaded with 4µg of Doxorubicin at increasing time points. The cells were washed 3 times with 1xPBS and lysed. The protein was precipitated with Silver Nitrate and centrifuged. The supernatant was collected and analysed for Doxorubicin by HPLC. Results suggest a slow increase in the drug localization of Doxorubicin from 1hr time point, releasing maximum at 4hr, in case of Lacto-MES. Lacto nanoparticles showed highest and sudden release of drug at 4hr point. Lacto-MES treated cells showed the presence of drug at 8hr time point but Lacto Nanoparticle showed no drug.

[0078] FIG. 43 shows Cell toxicity of surface modified nanoparticles (4300) according to the present invention. The 0.01*106 SupT1 and HL2/3 cells were seeded per well. Nanoparticles and MES were added at 500 µM concentration in triplicates. Cells were incubated with drug for 24hrs and the cytotoxicity was tested by MTT Assay. Result suggests that both MES and Lf-MES show low cytotoxicity at 500uM.

[0079] It will be apparent to those skilled in the art that various modification and variations can be made to the present invention without departing from the spirit and scope of the invention. There is no intention to limit the invention to the specific form or forms enclosed, but on the contrary, the intention is to cover all modification, alternative construction, and equivalents falling within the spirit and scope of the invention, as defined in the appended claims. Thus, it is intended that the present invention cover the modification and variation of this invention provided they come within the scope of the appended claims and their equivalents.
,CLAIMS:1. A novel nanoparticle formulation of biological molecules with or without drug for targeted delivery, includes:
taking, required biological molecules of appropriate material;
treating, said biological molecules with or without chemo-protective agents of appropriate materials derived from polymeric class for regulated release of active chemical entity;
mixing said protected biological molecule with one or more drug
adding, said mixture to rec. C-terminal apotransferrin (protein)/ apotransferrin/ C-terlactoferrin /lactoferrin;
incubating, total volume made between 200 and 500 µL with PBS (0.1 M, pH 7.4) on ice for 30 minutes;
slowly adding, protein-biological-drug mixture to 5 to 50 ml of olive oil at 4 °C with continuous dispersion by gentle vortexing;
sonicating, the sample at 4 °C using a narrow stepped titanium probe of ultrasonic sonicator;
freezing, the resulted mixture immediately at low temperature namely liquid nitrogen for 5 minutes;
transferring and incubating, said mixture on to ice and incubated for 4 hours;
pelletizing, formed particles by centrifugation at 8000 rpm for 15 minutes and the formed pellet was extensively washed with diethyl ether and dispersed in PBS and stored at 4 °C;
Said dispensed particles lyophilized with and without excipient and
wherein, from the prepared preparation the protein content in nanoparticles was estimated by Lowry method, Biological and drug molecules were estimated by chromatography.

2. The novel nanoparticle formulation according to claim 1, characterized to involve three steps; includes:
treatment of biological molecules (plasmid DNA, SiRNA, nucleic acid, antibody, protein, peptide, oligosaccharide, etc);
treatment of chemo-protective agent and mixing with chemotherapeutic agent; and
preparation of nanoparticles, wherein this procedure may be done with or without chemotherapeutic agent.

3. The novel nanoparticle formulation according to claim 1, characterized to use several biological molecules which are characterized spectroscopic and electrophoretic methods, biological molecules include but not limited to:
Plasmid; DNA; SiRNA; nucleic acid; antibody; protein; peptide and oligosaccharide; etc.

4. The novel nanoparticle formulation according to claim 1, characterized to contain and use native or recombinant complete or partial portion of apotransferrin or lactoferrin or transferrin nanoparticles.

5. The novel nanoparticle formulation according to claim 1, wherein apotransferrin or lactoferrin or transferrin nanoparticles are loaded with protected biological molecules with or without chemotherapeutic agent and characterized in the presence of several materials include but not limited to:
a means of taking lactoferrin loaded with biological molecules;
a means of Endosomal escape of lactoferrin loaded with biological molecules in presence of chemo-protective agent, polyamine oligomers etc., for site-specific localization;
a means of taking drug in combination of biological molecules loaded lactoferrin nanoparticles;
a means of taking drug and biological molecules with loaded lactoferrin nanaoparticles;
a means of regulated release of combination of drugs, drugs and biological molecules by crosslinkers based preparations; and
a means of taking surface modified drug, drug and biological molecules loaded lactoferrin nanoparticles with specific functional groups for specific target.

6. The novel nanoparticle formulation according to claim 1, characterized to deliver the composition in such a way that the time-delayed release of biological molecules with or without chemotherapeutic agent.

7. The novel nanoparticle formulation according to claim 1, wherein the characterization of nanoparticles are studied through one or more ways, including:
Morphological characterization (FE-SEM and AFM microscopy); Loading (DL) and Encapsulation efficiency (EE); Size-exclusion chromatography (SEC); Cell viability; Gel binding assay; MTT assay; Gel Retardation assay; Cell localization assay and pH release study.