Gene replacing therapy is a method of intervention of defective cistrons with the aid of familial engineering.It was done in worlds in the twelvemonth 1990 for familial diseases and is still at the developing phase. Gene replacement therapy helps to import foreign cistrons to alter the genotype of cells for the intervention of oculus diseases.In the cistron replacing therapy a wild opposite number of the impaired cistrons is tranfected into the appropriate cells and thereby reconstruct the production of the needed gene.This method is hard to practice.viral and nonviral methods are used to ease the transportation of cistrons into the cells.Transfer of cDNA version of a cistron under its abbreviated version of heterologic booster is done to acquire a dependable transfer.Because of many restrictions these cistrons are non able to restructure the endogenous look of the opposite number gene.There are some exceeding instances like that of the reaping hook cell anaemia in which the natural state type cistron look is regulated by the ? hematohiston booster and it is expressed usually.
1.2.2 Antisense therapy
Antisense therapy is a method of handling familial upsets or infections.Recently antisense has been widely used to track assorted type of malignant neoplastic diseases like lung malignant neoplastic disease, colorectal malignant neoplastic disease, pancreatic malignant neoplastic disease and many other diseases like asthma and arthritis. The chief rule involved in the antisense therapy is suppressing the interlingual rendition of a mark protein with the aid of complimentary oligonucleotide binding to the mark messenger RNA.
Genes are composed of dual coiling DNA.In order to specifically halt a cistron that is found to play a function in disease, its familial codification should be known.As a cistron gets turned on its familial codification in that section takes topographic point as a individual strand of RNA known as courier RNA.This courier RNA can be translated into a series of aminic acids to organize a protein, so they are called as sense sequence and the strand which is opposite in a Deoxyribonucleic acid dual spiral is called as the antisense strand.The antisense coding sequence of a disease cistron is used to do short Antisense DNAs.These antisense drugsceases the production of the disease doing protein by adhering themselves to messenger RNAs from disease cistrons so that familial codification in the RNA can non be read.GEM- 231, GTI- 2040, GEM -240 are some illustrations of antisense agents.
The antisense oligonucleotide when subjected into a cell hybridizes to the corresponding messenger RNA to organize a heteroduplex by Watson kink binding.As the semidetached house is formed suppression of the protein interlingual rendition coded by the sequence of edge Mrna takes place.Several mechanisms are involved in this suppression in which the most normally accepted mechanism provinces that the messenger RNA in the heteroplex is degradedby the omnipresent enzyme RNAase H.
1.2.3 RNAi Therapy
RNAi intervention is a station transcriptional cistron silencing in which a two-base hit stranded RNA causes specific dislocation of homologous Mrna sequences when it is introduced into the cell.RNA intervention has the capableness to let the negative ordinance of any gene.The stable propogation of the phenotype to progeny is enabled by transgene RNAi..RNAi therapy was first done in the invertebrate roundworm, caenorhabditis elegans.RNAi has been found in unicellular protozoons, insects, Fungis and in workss andvertibrates.RNAi Acts of the Apostless as a defense mechanism mechanism against viruses which include dual isolated DNA also.If double isolated Deoxyribonucleic acid is present in a cell, a ribonucleinase called the dicer identifies it and treat it into little dsRNA molecules.These dsRNA have 21 base pairs.These molecules are called as interfering RNAs.RNAi therapy is found to be an first-class manner for formalizing drug targets.RNAi therapy is chiefly done for patients holding diseases like malignant neoplastic disease, familial diseases and viral infections.The major defect of RNAi therapy is that the innate immune responses will be stimulated.Another major job in RNAi therapy is that there is ever competition with the endogenous RNA.
1.3.PROBLEMS ASSOCIATED WITH DELIVERY OF LARGE MOLECULES
Many failures occurred due to the systemic disposal of drugs in assorted clinical tests. This was due to the deficient sum of drugs making the mark site and besides taking to assorted side effects. In order to cut down the doses required and besides cut down he side effects drug aiming was required to present the drugs to the tissue of interest.The major defect of the big molecule bringing is the systemic toxicity and variable incursion of the molecules.An illustration for the bringing of big molecules into the oculus is the bringing of certain antibiotics into the aqueous wit of the oculus is prevented by the blood-aqueous barrier.Another barrier in the oculus is the blood-retina barrier which prevents the handiness of big molecular drugs into the posterior portion of the oculus which is made up of rtinal pigment epithelial cells and retinal capillaries. Large molecules can non go through through the blood-brain barrier and merely molecules holding a molecular weight of less than 400 John daltons can perfuse through it.Molecular therapeutics aid in the bringing of little molecular weight dugs into the encephalon where little protein lead drug molecules are put into force.
1.4 GENE DELIVERY METHOD.
The debut of healthy cistron into mark cells is termed as cistron delivery.It promote the look of normal protein and besides reconstruct right cellular function.It is a promising technique, Gene therapy has a polar function in the intervention of familial diseases.It is besides applicable to state of affairss which do non hold a familial component.Many different methods of cistron bringing are at that place and it can be chiefly subdivided into two groups
Viral cistron bringing
Non viral cistron bringing method
The cistron therapy vectors with sufficient aiming ability, transfection efficiency and safety must be achieved before cistron therapy could be used in man.The ideal cistron deliver vectors would hold any one of the undermentioned features, they are specificity, opposition to metabolic debasement, safety and an ability to show.
1.4.1 VIRAL GENE DELIVERY
Virus mediated cistron bringing is viral bringing method.A foreign cistron is packed into a viral atom and this virus inject the cistron inside a host cell.There are chiefly four types of viruses in usage of cistron therapy.They are adeno viruses, adeno associated viruses, lentiviruses, lentiviruses and reteroviruses.Among these Reteroviruses and adenoviruses are the most normally used vectors.
Adenoviruss are largely used in experimental and clinical attacks because of their advantages such as comparative easiness of production and wide mark tropism.These vectors were the first bioactive substances to be delivered to the bosom utilizing ultrasound targeted microbuble devastation method.
Adeno associated virus vectors are individual isolated Deoxyribonucleic acid viruses that can infect mark cells and may integrate their genome into the host cells.They have certain advantage when compared with adenoviral vectors which makes them superior in some clinical scenes demoing merely minimum immunogenicity, no direct toxicity.
Reteroviral Vectors are individual isolated RNA viruses that intergrate their genome into the host cell with the aid of contrary transcriptase.Thus it is non used in spliting cells and show the hazard of bring oning malignances by dysregulation of cistron at the extravagant intergration site.
Lentiviral vectors besides have the ability to incorporate their genome into the host cells.It is besides used for non spliting cells.It is based on the human immune lack virus type 1
Viral cistron bringing have several advantages, but besides have many unsought side effects such as viral toxicity and host immune rejection.
1.4.2 NON VIRAL METHODS
Non-viral methods include physical and chemical methods. The Physical methods such as microinjection, cistron gun, electroporation, sonoporation and magnetofection. The chemical methods include liposomes and miscelles.
126.96.36.199 SUPRAMOLECULAR SYSTEMS
Liposomes are microscopic pouch or bantam cysts made from man-made or natural fatty substances dwelling of phospholipids. Simply it is an aqueous compartment enclosed with same stuff as a cell membrane called phospholipids. The name liposome for these bantam bubbles came from two Grecian words, Lipos intending fat and soma significance organic structure. Liposomes were foremost described by British haematologist Dr Alec D Bangham FRS in 1961.
Since phospholipids are portion of the cell membrane they can easy perforate the horny cuticular bed of tegument and can pin down any substance that will fade out oil or H2O. The bilayer is formed by the agreement of hydrophilic groups confronting outside and hydrophobic dress suits repelled inwards, in this same manner the 2nd bed is formed by tail group repelled by the H2O inside the cell, in an acquous solution. Thickness of the bilayer is about 5-6nm Benefits of liposomes as drug carriers.The chief advantage of Liposomes are that it can be used to ensnare a broad assortment of hydrophilic and lipotropic drugs.Liposomes protect the drugs from enzymatic degradation.They have the capableness to ensnare a broad assortment of hydrophilic and lipotropic drugs.They Can be made with different sizes and composings.
Lipid monolayers with fatty acid nucleus and polar surface are called micelles. Inverted micelles are those with a Polar nucleus and fatty acerb surface. The procedure of organizing micelle is called micellization.By increasing the surfactant concentration ofa liquid the sum of surfactant adsorbed is increased at the liquid air and liquid container interfaces.When the concentration is further increased the surface assimilation of the surfactant molecules ends up with tightly packed monolayer.At this point interfacial tenseness become changeless and it reaches the majority solubility bound of the surfactant.If more surfactant is added to the solution it will tie in into little sums called miscelles.The concentration at which this happens is called critical miscellar concentration.The formation of miscelles is dependent on temperature and is normally measured at 25 degree celcius, .This temperature is called critical miscellar temperature.Depending on the concentration miscelles tend to alter their shape.At concentrations above critical miscellar concentration, it tends to be spherical and if the concentration is increased the form alterations to laminar or cylindrical.
In the instance of H2O indissoluble and meagerly soluble drugs can be solubilised with the incorporation of suited miscelles depending on the construction of the drug.Miscelles are besides used as drug bringing vehicles.Some of them are used as preservatives for drug preparations.
1.5 INTRACELLULAR TRAFFICKING
Endocytosis can be defined as procedure in which an extracellular molecule is invaginated into the cell organizing a membrane edge cyst around it.The part of the endosomes get invaginated into the cell organizing a a cyst enveloping the molecules known as endosomes.In the endocytic procedure the molecule which is to be ingested first gets invaginated and so it easy pinches to organize a cyst called as the endocytic cyst. The endocytosis can be divide into pinocytosis, phagocytosis and receptor mediated endocytosis
Pinocytosis is a type of endocytosis where the extracellular fluids are maily engulfed into the cells.They signifier little fluid filled cysts inside the cells.
Phagocytosis is a a type of endocytosis where molecules, chiefly solid atoms are engulfed into the cell.It besides helps in taking pathogens and other unwanted cellular stuffs.
Receptor mediated endocytosis
Receptor mediated endocytosis is where the engulfing of specific molecules takes place.Receptor proteins on the surface of the cell is responsible for the process.Uptake of low denseness lipoproteins into the cell is a authoritative illustration of receptor mediated endocytosis.
In endocytosis there are chiefly divided into early endosomes and late endosomes.
They are the cysts into which the invaginated molecules are foremost delivered. Early endosomes are besides known as screening endosomes.Rab4, Rab5 and reassigning receptor proteins are the characteristic proteins associated with early endosomes. The Rab proteins are distributed characteristically on the cell membranes.The early endosomes have an acidic environment inside it.
Rab5 is a protein that belongs to the Ras household of proteins. The rab proteins are involved in the trafficking of molecules into the cells and they are monomeric in nature.In the GTP edge form the Rab5 protein is active and in the GDP edge province they prove to be inactive.Rab5 proteins besides bind to other proteins known as Rab effecters to enable the moorage procedure in edocyotosis.Rab5A is found in the plasma membrane and clathrin coated cysts and Rab5c is found in early endosomes.
GREEN FLUORESCENT PROTEIN ( GFP )
Green fluorescent protein was foremost stray 30 old ages from a jelly fish Aequorea Victoria.After the GFP was cloned and expressed in other beings, led to the usage of GFP as a fluorescent protein marker.The importance of this protein led it to the Nobel Prize in 2008.Green fluorescent protein is a monomer with a conformation which is extremely stable.The stableness of GFP can be established by a half life tantamount to 26 hours in most of the cells.GFP expressed at 37 degree Celsius is far less fluorescent than GFP at 15 degree Celsius.The construction of GFP is similar to that of other proteins.The secondary construction is a series of pleated sheets and spirals due to the H bonding.The teriary construction is in the form of a barrel made up of 11 sheets and capped by helices.At the Centre there is a short concatenation of aminoacids which is the chromophore for the emanation of visible radiation. As shown in fig:1.
Fig:1 Tertiary construction of construction of GFP
GFP is made up of 238 aminic acids and with a weight of 27,000 atomic mass units.The fluorescence of eGFP is due to the chromophores which forms 65 to 67 ser-tyr-gly cyclisation of aminoacids.GFP has proved to be great tool with first-class potency in protein localization of function and cistron look.
Polymerase Chain Reaction ( PCR )
The speedy method of magnifying a peculiar piece of DNA in the testube is called polymerase concatenation reaction.With this method limitless transcripts of a individual Deoxyribonucleic acid molecules can be made from a mixture of deoxyribonucleic acid molecules.In the PCR technique the intended piece of DNA is amplified utilizing peculiar primers The chief construct behind this method is the ability of DNA polymerase to systhesise the DNA strand complimentary to its templet.
Deoxyribonucleic acid template-Targeted sequence of Deoxyribonucleic acid
Deoxyribonucleic acid polymerase-enzyme
Primer-short oligonucleotides complimentary to 3 ‘ terminal of each strand of Deoxyribonucleic acid to be amplified
Nucleotide-Deoxynucleotide triphosphates ( dATP, dCTP, dGTP, dTTP )
General stairss involved in PCR:
Choose the deoxyribonucleic acid to be amplified
Synthesise primer complimentary to the 3 ‘ terminal of DNA which need to be amplified
Heat the deoxyribonucleic acid sample and divide the strands and mix with the pimer
Binding of primer to complimentary sequence of DNA begind from 5 ‘ to 3 ‘ ( This polymerization reaction continues until each freshly synthesised strand identifies its ain primer
This consequence in the formation of DNA molecule indistinguishable to original molecule..
With the aid of an machine-controlled equipment more than a billion transcripts after 30 rhythms can be made.
PCR is used in the diagnosing of diseases like leukaemia and lymphoma.It is besides used in the designation of slowgrowing microorganisms.It is used in the sensing of viral DNA.Quantitative PCR methods are used to find the degrees of cistron expression.PCR fingerprint methods are used to place familial relationship between individuals.It is used in paternity testing and to find evolutionary relationships.
Sequencing is fundamentally needed for the Bio molecular researches. The sequencing Idahos done by the measure by measure incorporation of bases. sequencing by-synthesis is the technique which uses deoxyribonucleic acid polymerase enzyme to read the sequence of the templet at the 3? end point of an annealed primer strand with keen preciseness and to widen the primer by merely that base that is complementary to the templet base. If the designation of each base incorporated by the polymerase is possible so the sequence is easy read.
Incorporation of a concatenation ending nucleotide which is labelled fluorescently in individual nucleotide reactions uses the rule of. This method can besides be called as solid stage minisequencing and is used for multiplexed deoxyribonucleic acid sequences.
Transfection is the debut of an exogenic stuff ( DNA/RNA, Protiens or antibodies ) into a eucaryotic cell. The procedure is followed by the intergration of DNA later to its receiver cells chromosomal DNA. Usually these exogenic Deoxyribonucleic acid or RNA for transfection are isolated from a bacteriophage or a works, or animate being cell ensuing in complete reproduction of virus from which the familial stuff is isolated. But in eucaryotes non viral method is preferred.
1.6 ) Purpose
2. MATERIALS AND METHODS
2.1 ) MINI PREP
The bacterial cells were pelleted by microcentrifuging the nightlong civilization of MC 1061 Strain of bacteriums at 6000xg for 15 proceedingss and the supernatant liquid was removed.
The bacterial pellet was resuspended in 0.3ml of buffer P1 which is a combination of 50mM Tris Hcl Ph 8,10mM EDTA and 10µg/ml RNAase A.Lyse bluish reagent was besides added to the buffer P1.
To the above solution 0.3ml of buffer P2 which is a combination of200mM Na hydrated oxide, 1 % SDS in H2O was added and assorted exhaustively by inverting the sealed tubing a twosome of times.The solution turns bluish due to the presence of Lyse bluish reagent.
0.3ml of refrigerated buffer P3 which is 3.0M K ethanoate at Ph 5.5 was added to the above mixture and assorted exhaustively and incubated on ice and a precipitate of genomic DNA, cell dust and protein is obtained.The bluish coloring material disappears wholly.
Then the above suspension is microcentrifuged at 20,000xg for 10 proceedingss and the plasmid DNA incorporating supernatant liquid is removed.
The qiagen tip 2500 is equilibrated utilizing 1ml buffer QBT which is 750 millimeter Nacl,50mM MOPS at PH 7, 15 % isopropyl alcohol and 0.15 % of triton-100 and the column was allowed to empty.
The supernatant in the fifth measure was applied to the qiagen tip and was allowed to come in the rosin column.
The qiagen tip was washed with 2-2ml of buffer QC which is 1.0 M NACL,50mM MOPS at PH 7 and 15 % isopropyl alcohol.
The Deoxyribonucleic acid was eluted into polycarbonate extractor tubings utilizing 0.8ml of buffer QF which is 1.25M Nacl, 50 mM Tris-HCl at pH 8.5 and 15 % isopropyl alcohol.
The eluted DNA was precipitated out utilizing 0.7volumes of isopropyl alcohol at room temperature and it was microcentrifuged at 10,000xg for 30 proceedingss.
The pelleted DNA was washed with 7ml of 70 % ethyl alcohol and centrifuged at 10000rpm for 10 proceedingss.
The pellet was air dried and resuspended in sterile deionised H2O.
2.2 ) MEGA PREP
The bacterial cells were pelleted by centrifugating the nightlong civilization of LB at 6000xg for 15 proceedingss at 4 degree Celsius and the supernatant liquid was removed.
The bacterial pellet was resuspended in 50ml of buffer P1 which is a combination of 50mM Tris Hcl Ph 8,10mM EDTA and 10µg/ml RNAase A.Lyse bluish reagent was besides added to the buffer P1.
To the above solution 50ml of buffer P2 which is a combination of200mM Na hydrated oxide, 1 % SDS in H2O was added.The solution turns bluish due to the presence of Lyse bluish reagent.
50ml of buffer P3 which is 3.0M K ethanoate at Ph 5.5 was added to the above mixture and assorted exhaustively and incubated on ice and a precipitate of genomic DNA, cell dust and protein is obtained.The bluish coloring material disappears wholly.
Then the above suspension is centrifuged at 20,000xg at 4 degree Celsius for 30 proceedingss and the plasmid DNA incorporating supernatant liquid is removed.
The above supernatant liquid is once more centrifuged at 4 grades for 30 proceedingss at 20,000xg, and the supernatant liquid is obtained.
The qiagen tip 2500 is equilibrated utilizing buffer QBT which is 750 millimeter Nacl,50mM MOPS at PH 7, 15 % isopropyl alcohol and 0.15 % of triton-100 and the column was allowed to empty.
The supernatant in the 6th measure was applied to the qiagen tip and was allowed to come in the rosin column.
The qiagen tip was washed with 200ml of buffer QC which is 1.0 M NACL,50mM MOPS at PH 7 and 15 % isopropyl alcohol.
The Deoxyribonucleic acid was eluted into polycarbonate extractor tubings utilizing 35ml of buffer QF which is 1.25M Nacl, 50 mM Tris-HCl at pH 8.5 and 15 % isopropyl alcohol.
The eluted DNA was precipitated out utilizing 24.5ml of isopropyl alcohol at room temperature and it was centrifuged at 15,000xg for 30 proceedingss at 4 degree celcius.
The pelleted DNA was washed with 7ml of 70 % ethyl alcohol and centrifuged at 15000xg for 10 proceedingss at 4 degree celcius.
The pellet was air dried and resuspended in sterile deionised H2O.
2.3 ) POLYMERASE CHAIN REACTION ( PCR )
Change by reversal primer
Nuclease free H2O
GREEN TAQ ( µl )
OLIGO-110 ( µl )
OLIGO-113 ( µl )
OLIGO-111 ( µl )
OLIGO-112 ( µl )
TEMPLATE ( µl )
NUCLEASE FREE WATER ( µl )
In the first tubing 50µl of green taq,1.25 µl of oligo 110,1.25 µl of oligo 113, 1 µl of templet and it was made upto 100µl with 46.5 µl of H2O.
In the 2nd tube50 µl of green taq,1.25 µl of oligo 110,1.25 µl of oligo 113, 1 µl of templet and it was made upto 100µl with 46.5 µl of H2O.
The samples were placed in the pcr machine and the palpebra was closed.
The plan was set for 2 kilobits and the annealing temperature was set at 45 degree celcius and the pcr machine was allowed to run.
2.4 ) AGAROSE GEL ELECTROPHORESIS
Gel casting trays
Electrophoresis buffer which is Tris ethanoate EDTA
Loading buffer which was 1 % glycerin
1 % agarose gel was prepared with 0.5µg/ml of ethidium bromide at 60 degreee celcius and it was poured into projecting trays.
Then the sample combs were placed closer to one terminal of the tray and it was allowed to solidify.
The combs were so taken out without upseting the Wellss formed.
Then the tray was placed on the cataphoresis chamber with the good near to the negative electrode because DNA are negatively charged.
Then Tris Borate EDTA buffer was added to the cataphoresis chamber until it covers the top of the gel.
The 6µl of the sample assorted with 2 µl of the lading dye were loaded into the Wellss without puncturing the Wellss.
At either side of the sample wells ladders were besides loaded which is composed of chromatographically purified 15 DNA fragments in base brace.
Then the palpebra was closed and the cords were plugged and the power was switched on and the electromotive force was set to 120V.
It was allowed to electrophorese for 40 proceedingss.
Then the casting tray was placed in the transilluminator and the sets were observed under the U.V. and the imagination was done.
2.5 ) TRANSFECTION
vero cells were incubated in the medium with 2ml TxR-BSA at a concentration of 10mg per milliliter and 2ml of rab5-GFP at a concentration of 2mg per milliliter. Then cells were washed in 3times in unfertile PBS, so and the cells were incubated for 4 hours.
The cells are fixed on coverslips before the Microscopy.
Three screen faux pass were taken in three separate dishes and the screen faux pass were washed with PBS three times.Then The cell civilization was placed on the screen faux pass and washed three times with phosphate buffer solution and incubated for 16 hours before arrested development.
METHANOL FIXATION FOR LAMP
100 % methyl alcohol was poured onto the cells in dish where the screen home bases are placed and the dish was left on the bench for 5 proceedingss, so wash repeatedly with PBS.
2 % Para formaldehyde solution readying.
1g paraformaldehyde, 5ml phosphate buffer solution made upto 10 times with water,1ml of 5N NaoH and 1ml of 5N Hcl was assorted together to fix paraformaldehyde solution for aldehyde arrested development.
Aldehyde arrested development for EEA1 and Transferrin.
The cells were rinsed cells with PBS at room temperature. 2 % paraformaldehyde in PBS was poured into the dishes for aldehyde arrested development and keptfor 15 min at room temperature.The home bases were washed 3times with PBS before permeablisation.The cells were permeabilized with 0.1 % Triton X-100 in PBS for 5 minutes.Then the dishes were rinsed with PBS.
2.6 ) MICROSCOPYThe fixed cells were imaged utilizing a microscope connected to a camera ( Nikon ) and it was read into the computing machine screen.
3 ) Consequence
The plasmids were synthesisised utilizing the Megaprep and 1.9g of the plasmid was obtained.The output was obtained utilizing a U.V. spectrophotometer.After the gel agarose cataphoresis straight sets were obtained which showed the plasmids were additive and the sets were matching to the molecular weight of the plasmids.The mention was the ladder on either side of the sample Wellss.
PCR was done with the plasmids and a proper rab5 gfp sequence was obtained.The consequences osf the sequencing informations showed the sequence as that of the rab5-gfp.
Ladder tube 2 tubing 1 ladder
51Rab5a-111: Oligo 111
51Rab5a-113: Oligo 113
51Rab5a-110: Oligo 110
51Rab5a-112: Oligo 112
Sequencing was done and the sequencing information was analysed and the sequence was read.The sequence was read and the GFP-Rab5 sequence was read in the sequence.
After the sequencing consequences were obtained the plasmids along with bsa Texass red were transfected into the vero cells and the fluorescent microscopy was done.and the consequences obtained showed that in the early sorting endosomes the gfp rab5 and the EEA1 carbon monoxide localises, where as in the late endosomes it ne’er happened and the green and the ruddy fluorescence showed individually in those images.In Fig:2 it clearly shows the ruddy coloring material of BSA Texas ruddy and the green coloring material of GFP-Rab5 separately.In Fig: 3 it shows that the GFP-Rab5 is carbon monoxides localised with Texas-Red in a individual cyst.
Gfp-Rab5 led to understanding the nature of cyst formation in the cells and about early endosomes.In the plasmids synthesised the sequence informations obtained showed that it was the rab5 gfp plasmid sequence.Then in the transfection consequences enabled to understand and place that that the rab5 and EEA1 colocalised with the early sorting endosomes.In the late endosomes the flouresence were seen individually and it distinguished the early endosomes from the late endosomes.
Through this we were able to supervise the destiny of these polymers in the cell and was able to track them in different compartments utilizing fluorescent probes.The imagination of both unrecorded and fixed cells is possible utilizing rab5-gfp.Since the compartments are different in their features inside the cells and the consequences showed that EEA1 merely corresponds to early endosomes, LAMP to late endosomes and Transferin to lysosomes. The challenge of bringing of the molecules particularly the macromolecular drugs can be accomplished by the intramolecular trafficking.