Protein and Peptide Drug Delivery - A Brief Review

 

L. Srinivas*, V. Manikanta, M. Jaswitha

GITAM Institute of Pharmacy, GITAM Deemed to be University, Rushikonda, Visakhapatnam-530045

 

ABSTRACT:

Peptides and proteins are complex architecture therapeutics useful in several diseases. Several pharmaceutical and biopharmaceutical challenges limit their clinical applications. Continuous efforts are focussed for formulation of this therapeutics into safe and effective delivery systems. The present review briefly describes the possible methods for the delivery of protein and peptide drugs through various routes.

 

 

 

INTRODUCTION:

Protein and peptides are increasingly recognised as potential candidates for the development of new therapeutics for variety of human ailments. Due to their relatively specific mode of action, proteins and peptides can be administered at comparatively low doses for therapeutic effects. This potent therapeutics is indicated for several chronic conditions such as cancer, hepatitis, diabetes, rheumatoid arthritis and leukaemia^[1]. There are several protein and peptide drugs which are approved by the FDA listed in Table 1.

 

The advent of biotechnology and modern analytical tools has promoted discovery and large scale production of protein and peptide drugs. Peptide is a short chain of amino acid residues with a defined sequence (e.g. Leuprolide). Protein is polypeptides which occur naturally and have a defined sequence of amino acids and a three-dimensional structure (e.g. Insulin). Parental administration is the major route for administration of therapeutic proteins and peptides^[2]. Major barrier for non-invasive administration is permeation across a cell layer. The large molecular size, charge, hydrophilic nature and low stability also contribute to their poor bioavailability. Therefore, there is a continuous research for the development of formulation for the delivery of protein and peptide drugs^[3].

 

Fig. 1: Structure of protein

 

Table 1: FDA approved protein and peptide drugs^[4]

S. No.	Therapeutic area	Protein drugs
1	Immunology	Belimumab, Belatacept, Metreleptin, Siltuximab, Vedolizuma, Peginterferon beta-1a, Secukinumab, Ixekizumab, Daratumumab.
2	Dermatology	Ipilimumab, Pembrolizumab, Nivolumab, Secukinumab, Ixekizumab.
3	Oncology	Ipilimumab, Bretnuxiamab Vedotin, Asparaginase Erwinia Chrysanthemi, Pertuzumab, Ziv-afilbercept, Tbo-filgrastim, Ado-trastuzumab emtansine, Obinutuzumab, Ramucirumab, Pembrolizumab, Blintumomab, Nivolumab, Filgrastim-sndz, Dinutuximab, Daratumumab, Elotuzumab, Atezolizumab.
4	Nephrology	Belatacept, Glucarpidase, Daclizumab.
5	Haematology	Bretnuxiamab vedotin, Asparaginase erwiniachrysanthemi, Tbo-filgrastim, Obinutuzumab, Siltuximab, Blintumomab, Parathyroid hormone, Idarucizumab, Daratumumab, Coagulation factor IX recombinant human, Antihemophilic factor, Coagulation factor XIII A subunit, Coagulation factor IX, Antihemophilic factor, C1 esterase inhibitor recombinant, antihemophilic factor porcine, B-domaintruncated recombinant, Coagulation factor IX, Antihemophilic factor, Von Willebrand factor.
6	Ophthalmology	Afilbercept, Ocriplasmin.
7	Endocrinology	Taliglucerase alfa, Albiglutide, Dulaglutide, Parathyroid hormone.
8	Gastroenterology	Taliglucerase alfa, Ziv-afilbercept, Ramucirumab, Vedolizumab.
9	Obstetrics	Pertuzumab, Ado-trastuzumab Emtansine
10	Gynaecology	Pertuzumab, Ado-trastuzumab Emtansine
11	Infections and infectious disease	Raxibacumab, Obiltoxaximab
12	Musculoskeletal	Golimumab injection for IV use, Tocilizumab, Elosulfase alfa, Peginterferon beta-1a, Infliximab-dyyb, Daclizumab.
13	Rheumatology	Golimumab injection, Tocilizumab, Infliximab-dyyb, Etanercept-szzs
14	Genetic disease	Elosulfase alfa, Asfotase-alfa, Sebelipase alfa
15	Endocrinology	Albiglutide, Dulaglutide, Parathyroid hormone
16	Paediatrics	Dinutuximab, Asfotase-alfa
17	Neonatology	Dinutuximab, Asfotase-alfa
18	Cardiology	Alirocumab, Evolocumab, Sebelipase alfa
19	Vascular diseases	Alirocumab, Evolocumab, Sebelipase alfa
20	Pulmonary disease	Mepolizumab, Necitumumab.
21	Respiratory disease	Mepolizumab, Necitumumab, Rreslizumab

 

STRUCTURE OF PEPTIDES AND PROTEINS:

The structure is as shown in Fig.1 contains a primary structure of amino acids individual arrangement, secondary coiled α-helix and pleated sheets, tertiary three dimensional arrangement and quaternary association of ternary forms^[5]

 

STABILITY OF PROTEINS:

Proteins are only marginally stable under physiological conditions. Forces such as hydrophobic, electrostatic interactions and hydrogen bonding act more as stabilizing factors. Protein degradation pathways such as chemical, physical and biological as showed in Fig. 2 presents a challenge to formulation scientists, for the development of stable pharmaceutical preparations. The measures to improve chemical and physical stability are summarised in Table 2. Biological stability can be improved by co-administration of enzyme inhibitors or by altering 3D structural orientation.

 

 

Table 2: Stability aspects of proteins and peptides

S. No.	Stability	Problem	Overcome /Prevention
1	Chemical Instability
A	Deamidation	Spontaneous degradation and loss of amino acid sequence homogeneity Denaturation, increase the immunogenicity	Buffer composition, lowering of Ph
B	Oxidation	Air, residual peroxide content, or intense fluorescent light, may lead to the decomposition of protein and peptide	Lyophilisation, Use of antioxidants, chelating agents, protection from light.
C	Proteolysis	Exposure to Harsh conditions such as high pH, high temperature or proteolytic enzyme	Storing at cold and sterile conditions
D	Disulfide exchange	It alters its 3 dimensional structures and results in its biological activity.	By thiol scavengers such as P- mercuribenzoate, N-ehylmaleimide, Copper ions.
D	Racemisation	Racemisation may form peptide bonds that are sensitive to proteolytic enzymes.	Addition of thiol scavengers such as P- mercuribenzoate, N-ehylmaleimide, Copper ions, may prevent susceptible sulphur and disulphide.
E	β-elimination	β-elimination of cystine residue leads to destruction of disulphide bonds of protein, due to high temperature.	Blocking thiol group
2	Physical Instability
A	Aggregation and precipitation	Ionic complexation, salting out, charge neutrality close to the isoelectric pH, and results in limiting solubility of the molecule. Increase in thermal motion of the molecule due to agitation.	Use of nonionic surfactants to prevent aggregation and sugars to stabilise the proteins
B	Denatured protein	It may lead to the decrease in solubility, alteration in surface tension, loss of crystallizing ability, changes in constituent group reactivity and molecular profile, vulnerability to enzymatic degradation, loss of antgenicity and loss of specific biological activity.	Maintaining pH, ionic strength and temperature.
C	Adsorbed protein	If the peptide and protein drug entities are adsorbed at interface there may reduction in concentration of drug available to show its function	Use of Surfactants, smooth glass walls and reduces in excess agitation.

 

 

Fig. 2: Degradation pathways of proteins.

 

PROTEIN DELIVERY:

The formulation design and delivery of protein drug delivery involves not only protection of  protein/peptide from enzymatic degradation but also aid in enhancing its absorption without altering in biological activity. Hence there are several techniques involved for the successful delivery of proteins and peptides for their specific site of action.

 

INJECTABLE DOSES:

Parental route is the most efficient way for the delivery of proteins and peptides.The parenteral preparations generally contain suitable solvents and buffers in order to solubilise and stabilize the drug. Further the formulations are subjected to freeze drying to improve the stability during storage. To overcome the problems of denaturation; the formulation is designed with excipients such as polyalcohols and polymers to protect them during freeze drying and storage. Some protein and peptide drugs administered through various parenteralroutes are given in Table 3.

 

Table 3:  Protein and peptide drugs by parenteralroute^[6]

S. No	Protein/ Peptide	Route of administration	Applications
1	Ziv-aflibercept	Intravenous infusion	Metastatic colorectal cancer
2	Ocriplasmin	Intravitreal injection	Symptomatic vitreomacular adhesion
3	Raxibacumab	Intravenous infusion	Inhalational anthrax
4	Belimumab	Intravenous infusion	Systemic lupus erythematosus
5	Ipilimumab	Intravenous infusion	Unrespectable or metastatic melanoma
6	Belatacept	Intravenous infusion	Prophylaxis of organ rejection (kidney transplant)
7	Bretnuxiamab vedotin	Intravenous infusion	Hodgkin lymphoma and systemic anaplastic large cell lymphoma
8	Asparaginase Erwiniachryanthemi	Intramuscular injection	Acute lymphoblastic leukaemia
9	Afibercept	Intravitreal injection	Neovascular (wet) age related macular degeneration (AMD), Macular edema following central retinal vein occlusion (CRVO)
10	Velaglucerase alfa	Intravenous infusion	Type 1 gaucher disease
11	Tesamorelin	Subcutaneous injection	Lipodystrophy
12	Tocilizumab	Subcutaneous injection and intravenous injection	Rhemutatoid and systemic juvenile idiopathic arthritis
13	Collagenase clostridium histolyticum	Intralesional injection	Dupuytren’s contracture
14	Alglucosidase alfa	Intravenous infusion	Pompe disease
15	Denosumab	Subcutaneous injection	Postmenopausal osteoporosis
16	Incobotulinumtoxin A	Intramusucular injection	Cervical dystonia
17	Pegloticase	Intravenous infusion	Chronic gout
18	Etanercept	Subcutaneous injection	RA, psoriatic arthritis, plaque psoriasis, ankylosing spondylitis[7]
19	Salmon calcitonin	IM, SC., intranasal	Osteoporosis[8]
20	Pegfilgrastim	Subcutaneous injection	Neutropenia[7]
21	Bivalirudin	Intravenous infusion	Anticoagulant [9]
22	Desmopressin	IV, IM, SC, intranasal	Nocturnal enuresis [10]

 

EMULSIONS/MICROEMULSIONS:

Protein drug delivery through emulsions and microemulsions is one of the promising approach in biomedical, pharmaceutical, and cosmetics. Delivery of influenza vaccine and diphtheria toxoid, BSA in emulsions is the proven examples. They offer wide range in applications such as protection, permeation and prolongation of drug action^[11-13].

 

Various methods have been utilized to formulate proteins, such as coacervation/desolvation, thermal gelation, emulsification, self-assembly, and solid-in-oil dispersions^[14-17]. The formation and stabilization of the emulsions depends on droplet size and emulsifying agent^[18]. Recent reports on delivery of insulin as water-in-oil emulsion by phospholipid-based anhydrous reverse micelles for oral delivery of peptides have been reported^[19]. Current research in microemulsion delivery of proteins and peptides are shown in Table 4.

 

Table 4: Various proteins/peptides used in Microemulsions

Protein/ Peptide	Composition				Applications	Reference
	Aqueous phase	Oil phase	Surfactants	Co-surfactants
HIV transactivator protein TAT (TAMRA-TAT)	Water	Miglyol 812	Capmul MCM	Tween 80	Antiviral	[20]
Insulin	Water	Isopropyl myristate or oleic acid	Tween 80	Isopropanol	Diabetes Type-I	[21]
P42 Peptide	Water	TAMR	The lipid mixture Aonys	---	Huntington Disease	[22]
Ovalbumin (Ova) and Quil A	Water, ethanol	Isopropyl myristate	Capryl-caprylyl glucoside (CCG)	Lecithin Model	antigenic vaccine	[23]
Lidocaine	water, ethanol	Olive oil	Migloyl	Lecithin	Local anaesthetic	[24]
Bluetongue virus serotype 4 inactivated suspension	Inactivated virus suspension	Isopropyl myristate	α-hidroxy-whidroxypropyloxyethylen)- poly (oxypropylen) poly (o xyethylen)	Polysorbate 80	Antiviral	[25]
Aprotinin	0.9% NaCl	Isopropyl myristate and oleic acid	Labrasol and CR	Ethanol and Isopropanol	In pancreatitis therapy as a protease inhibitor	[26]
Insulin	Phospholipid dispersion with buffers	Glyceryl monooleate (GMO), Tween 20, and polyethylene glycol (PEG 400)	Tween 20	---	In Hyperglycaemia	[27]

 

 

 

 

ENZYME INHIBITOR:

Presystemic metabolism of proteins and peptide drugs can be reduced by co-administration of enzyme inhibitors. Use of proteases and peptidases inhibitors such as soybean trypsin inhibitor, FT-448, Bestatine, Comostate amylase, Leupeptine, Aprotinin^[28]. Furthermore, Bacitracin, Amastatin, Boroleucin and Puromycin^[29] have been used to avoid enzymatic degradation of drugs such as Leucine enkephalin^[30] and human growth hormone^[31][32] (Table 5).

 

 

Table 5: Protein and peptide delivery through co-administration of Enzyme inhibitors.

Enzyme inhibitor	Molecules inhibited	Effect on peptide drugs	Reference
Soybean trypsin inhibitor, FK-448	Chymotrypsin	Enhanced intestinal absorption of insulin in rats and dogs. Suppressed digestion of insulin by pancreatic enzymes	[33]
Aprotinin	Serine proteases, specifically trypsin, Chymotrypsin, and plasmin	Intraileally administered insulin with aprotinin led to decrease in blood glucose of 30% compared with controls	[34]
Puromycin	Serine and metallopeptidases	Improved stability of Leucine enkephalin, and stability and permeability of d-Ala2, d-Leu5 enkephalin (DADLE)	[35,36]
N-acetylcysteine	Inhibits amino peptidase N and has mucolytic properties	--	[37,38]
Bacitracin	Trypsin and Pepsin, amino peptidase N	Used to increase delivery of Insulin, Met-kephamid and Buserelin	[39]

 

MICRO PARTICLES:

Microparticles offer effective encapsulation, controlled and targeted delivery of drugs. Delivery of protein and peptide drug by using Microparticles as carriers has proven application for delivery of vaccines (eg: Hepatitis B Vaccine etc) and diagnostic agents (Table 6). The pH response of the microspheres promotes oral delivery by protecting the drugs from proteolytic enzymes^[40].

 

Table 6: Microparticle deliveryof proteins^[41]

Active ingredient	Product Name	Therapeutic application
Leuprolide	Lupron Depot (1989)	Palliative treatment of advanced prostatic cancer
Octreotide	Sandostatin LAR (1998)	Acromegaly and endocrine gastroenteropancreatic tumour
Somatropin	Neutropin depot	Growth disorder in paediatric patients
Triptorelin	Trelstar depot	palliative treatment of advanced prostatic cancer
Abarelix	Plenaxis	palliative treatment of advanced prostatic cancer

 

PENETRATION ENHANCERS:

Permeability is one of the major hurdles for protein and peptide delivery through mucosal transport by passive diffusion due to their large molecular size. Several approaches have been reported to enhance permeation through skin, oral mucosa, intestinal cell membrane, nasal mucosa etc. Use of surfactants, bile salts, hydrophilic polymers, cyclodextrins, dihydrofusidates, cheating agents, oleic acid, dimethyl sulphoxide are reported to enhance permeability of protein drugs through various route^{[42- 44]}.

 

Recently, co-administration of cell-penetrating peptides with therapeutic peptides has been attempted in order to increase absorption of the therapeutic action. Forexample: Insulin is co-administered with CPPs consisting of six to ten repeats of Arginine led to increase GI uptake of Insulin^[45]. A novel absorption enhancer, viz. Zonula occludens toxin (ZOT)^[46], chitosan^[47], thiolated polymers^[48] and Pz-peptide have all been studied as penetration enhancers for oral insulin delivery, and have resulted in effective reduction of glucose levels in the body^[49].

 

IONOTOPHORESIS:

Principles of electro repulsion and electro osmosis are used for ionotophoretic delivery of charged and uncharged delivery of peptides respectively^[50]. LidoSite® is a FDA approved ionotophoretic system for delivery of Lidocaine^[56]. Several other ionotophoretic  systems are under pipe line research  for delivery of proteins such as Insulin, TRH, Salmon Calcitonin, Delta sleep inducing peptide, LHRH, Vasopressin, Leuprolide, gonadotropin releasing hormone etc.^[51-55]

 

NANOCARRIERS:

Nanocarriers have gained considerable attention as colloidal carrier systems for the delivery of anticancer drugs. Protein Nanocarriers possess various advantages including their low cytotoxicity, abundant renewable sources, high drug binding capacity and significant uptake into the targeted tumour cells. Some of the protein based nanocarriers are listed below (Table 7)

 

Table7: Different Nanocarriers for delivery of proteins.

Protein	Carrier systems	Reference
Insulin	Nano-cubicles	[57]
Insulin, calcitonin, HGF (Humangranulocyte colony stimulating factors)	Nanocapsules	[58]
Salmon calcitonin	PLGA-nanoparticle	[59]
Insulin	Acrylic-based co-polymer nanoparticle	[60]
Cyclosporine	Lipid microemulsions	[61]
Leucine encephalin	Sugar coupling with cellobiose and gentiobiose	[62]
Insulin	Chitosan nanoparticle	[63]
HIV Protease (CGP57813)	pH sensitive nanoparticle	[64]
DGAVP	Niosomes	[65]

 

LIPOSOMES:

Liposomes demonstrate great potential as a carrier system for systemically administered protein therapeutics (Table 8). The aqueous core of liposome preserves the structure and conformation of protein, while the lipid court helps to improve absorption across biological membranes. If constructed from biocompatible and biodegradable materials, liposomes cause very little to no antigenic, pyrogenic, allergic or toxic reactions^[66].

 

Liposomes as carriers can be delivered through different routes such as parenteral, oral, buccal, pulmonary, intranasal, ocular, transdermal. DepoFoam liposomal formulations of a protein such as insulin and peptides such as Leuprolide, Enkephalin and Octreotide have been developed and characterized.  Some of the clinically approved liposomal based drugs are liposomal Amphotericin, liposomal doxorubicin. The recent advances in oral delivery of peptides are the wheat germ agglutinin-carbopol-modified liposome^[67].

 

PEG-grafted liposomes have increased circulation time, reduced aggregation and decreased capture by the RES. PEGylated liposomes, or Stealth™ liposomes (Johnson & Johnson, NJ, USA), have been used to deliver the anthracycline chemotherapeutic doxorubicin and were able to deliver preferentially to the tumour site, likely via the enhanced permeability and retention (EPR) effect^[68].

 

Table 8: Liposomal delivery of proteins and peptides

Sl. No	Protein /peptides	Production procedure	Applications/functions	Reference
1	Adamantyltripeptides	Dry lipid hydration	Treatment for viral diseases, tumours andimmunomodulations	[69]
2	Anti-Ovalbumin antibodies	Dry lipid hydration	Antibodies	[70]
3	Basic fibroblast growth factor	Freeze-thawing extrusion	Cardio protective	[71,72]
4	Bovine serum albumin	Reversal evaporation Double emulsification Freeze-thawing	Biochemical applications including ELISA (Enzyme-Linked Immunosorbent Assay), immunoblots, and immunohistochemistry	[73,74]
5	Calcitonin	Dry lipid hydration	Hypercalcemia or osteoporosis	[75,76]
6	Enkephalin	Double emulsification	Pain modulation.	[77]
7	Epidermal growth factor receptor	Freeze-thawing extrusion	Drug targeting	[78]
8	Haptides	Double emulsification	Tissue regeneration, soft tissue augmentation, skin repair, and wound healing	[79]
9	Haemoglobin	Dry lipid hydration extrusion	Carries oxygen	[80,81,82,83]
10	Horseradish peroxidise	Extrusion	It act as conjugate to determine the presence of a molecular target.	[84]
11	Human gamma-globulin	Dehydration-rehydration	Hepatitis	[85]
12	Insulin	Reverse phase evaporation,Freezing-thawing	Antidiabetic	[86,87]
13	Leishmania antigen	Freezing-thawing extrusion	Leishmania infection	[88]
14	Leridistim	Double emulsification	Local anaesthetics’, anticancer drugs, analgesics.	[89]
15	Leuprolide	Dry lipid hydration, Reverse phase evaporation	Prostate cancer, endometriosis, uterine fibroids, central precocious puberty	[90,91,92,93]
16	Nerve growth factor	Reverse phase  evaporation	Dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimia nervosa.	[94]
17	Octreotide	Double emulsification	Tumours, Bleeding oesophageal varices, Radio labelling, Hypoglycaemiain neonates	[95]
18	Progenipoietin	Double emulsification		[96]
19	Recombinant malaria protein antigens	-	Vaccine	[97]
20	Superoxide dismutase	Dehydration-rehydration, Dry lipid hydration Pro liposome	Chronic inflammation in colitis,  inflammatory bowel disease	[98]
21	twin-arginine translocation (TATA binding protein)	Extrusion	Elisa, protein array, western blot	[99]

 

SOLID LIPID NANOPARTICLES:

Development of solid lipid nanoparticles is one of the emerging fields of the lipid nanotechnology with several potential applications in drug delivery; clinical medicine and research (Table 9)^[100]. SLNs reduce mobility for incorporated drugs, which is a desirable feature for controlled drug release. The diameter varies between 50nm to 1µm and they can be stabilized using nontoxic surfactants, polymers or both ^[101].

Peptides such as Cyclosporine A, Insulin, Calcitonin and Somatostatin have been introduced through this technique^[102]. SLNs are also well suited to load synthetic liphophilic drugs like Etracaine, Etomidate and Prednisolone for prolong release at the site of action^[103]. SLNs prepared by High pressure homogenization (HPH) technique loaded with Tamoxifen a nonsteroidal anti oestrogen used in hormones positive early breast cancer and their antiproliferative activity was studied in vitro in the Michigan Cancer Foundation-7(MCF -7) cell line ^[104]. SLNs stabilized with tristearin enhance the half life and mean residence time in plasma of the anticancer drug Tamoxifencitrate^[105].

 

Table 9: Different types of protein/peptide based solid lipid nanoparticles

Sl.No	Peptide/ Protein	Method of preparation	Applications/Functions	References
1	BSA	Adsorption onto SLN	Biochemical applications including ELISA (Enzyme-Linked Immunosorbent Assay), immunoblots, and immunohistochemistry	[106]
2	Calcitonin	Solvent evaporation (w/o/w)	Hypercalcemia or osteoporosis	[107]
3	CyA	HPH hot dispersion	Enhanced ocular retention time on the cornea surface	[108]
4	CyA	HPH cold dispersion	Enhanced ocular retention time on the cornea surface	[109]
5	CyA	Warm microemulsion (o/w)	Enhanced ocular retention time on the cornea surface	[110]
6	Gonadotropin	Solvent displacement	Primary hypothalamic amenorrhea, hypogonadotropic hypogonadism	[111]
7	Insulin	Solvent evaporation (w/o/w)	Increases stability and acts as anti-diabetic	[112]
8	Insulin	Warm microemulsion	Oral delivery	[113]
9	Insulin	Solvent displacement	Oral delivery	[113]
10	Insulin	Supercritical CO2 (PGSS)	Oral delivery	[114]
11	HSA	Adsorption onto SLN	Transports thyroid hormones	[115]
12	[D-Trp-6] LHRH	Warm microemulsion (w/o/w)	Breast cancer	[116]
13	Lysozyme HPH	cold dispersion	Vaccine	[117]
14	Ovalbumin	Melt-dispersion (o/w)	-	[118]
15	Thymopentin	Warm microemulsion	Immunostimulant	[119]

 

POLYMERIC NANOPARTICLE:

Polymeric nanoparticles are nanosized colloidal materials able to encapsulate, adsorb or covalently bind drugs. Since most polymer properties can be easily modified, nanoparticle constitutes a versatile drug delivery system^[120].

 

In order to improve the oral bioavailability of proteins and peptides, new range of biodegradable polymeric nanoparticle has been used which can enhance the stability, control the release and the pharmacokinetic parameters^[121]. They are also known to show reduced toxicity in the peripheral healthy tissues^[122]. Polymeric nanoparticles are also helped in delivery of protein drugs like BSA and (HSA) Human serum albumin^[123,124].

 

Many methods have been developed or adapted for the preparation of polymer nanoparticles. Most of these methods include two main steps: preparation of an emulsified system followed by nanoparticles formation. The latter step can be achieved by polymer precipitation or gelation or by monomer polymerization^[125]. A list of the most widely used polymers is listed below in Table 10^[126].

 

 

Polymeric nanoparticles also help in ocular delivery of certain drugs like calcitonin, and enkephalin by systemic absorption^[127-129]. Calcitonin (Miacalcin), Desmopressin (DDAVP), Nafarelin acetate (Synarel), calcitonin, salmon (Fortical), and Oxytocin (Syntocinon) are few of the marketed proteins and peptidesfor nasal administration^[130] (Table 11).

 

Table 10: Different polymers used for nanoparticles^[131]

Materials	Examples
Synthetic homopolymers	Polylactide, (PLA), Poly(lactide-co-glycolide) ( PLGA), Poly(ε-caprolactone) (PCL) ,Poly (isobutylcyanoacrylate) (PICBA),Poly(isohexylcyanoacrylate )( PIHCA) , Poly (n-butylcyanoacrylate) ( PBCA), Polyacrylates and polymethacrylates
Natural polymers	Chitosan, Alginate, Gelatine, Albumin
Co polymers	Polylactide-poly (ethylene glycol) (PLA-PEG), Poly(lactide-co-glycolide)-poly (ethylene glycol) (PLGA-PEG) , Poly (ε-caprolactone)-poly (ethylene glycol) ( PCL-PEG),Poly(hexadecylcyanoacrylate-co-poly(ethylene glycol) cyanoacrylate) (Poly(HDCA-PEGCA))
Colloid stabilizers	Dextran,Pluronic F68,Poly(vinyl alcohol) (PVA),Copolymers (see above),Tween® 20 or Tween® 80

 

 

 

 

 

 

 

Table 11: Protein and peptide delivered through polymeric nanoparticles. ^[132-147]

Protein/peptide	Polymer (*)	Applications
Bone morphogenetic protein	PLA-PEG copolymer (650Da PLA-200Da PEG)	Bone formation
Transforming growth factor-β	50:50 PLGA (40-100 kDa)(+demineralised bone matrix)	Bone formation
Throtropin	75:25 PLGA (11 kDa)	Central nervous system dysfunction
Growth hormone releasing factor	75:25 PLGA (91 kDa)	Growth hormone deficiency
Somatostatin Analogue	55:45 PLGA (23-76 kDa)	Acromegaly, tumours
Neurotensin Analogue	PLA (2-6 kDa)	Psychotropic
Cyclosporine A	50:50 PLGA (0.44 & 0.80dL/g)	Immunosuppressant
Colonizing factor antigen (E. coli)	PLGA(0.73Dl/g)	Oral vaccine
Cholera toxin B subunit	PLA (2 kDa)	Oral vaccine
Diphtheria toxoid formalin treated	PLA (49 kDa)	Vaccine
Ovalbumin	50:50 PLGA (22kDa),85:15 PLGA (53kDa)	Vaccine
Tetanus toxoid	50:50 PLGA (100kDa)	Vaccine
LHRH antagonists	50:50PLGA,75:25PLGA	Tumour suppression
Horse radish peroxidise, Bovine serum albumin	75:25PLGA (10kDa)	Marker proteins Mechanistic studies
Leuprolide	PAA	Prostatecancer.
Insulin	Thiolated PAA	Blood sugar reduction

 

AQUASOMES:

Aquasomes are self-assembled nanoparticle carrier system useful for delivery of protein pharmaceuticals, enzymes and antigens^[148]. They act as a carrier through a non-covalent bonding and good stability^[151]. Several reports have shown the delivery of Aquasomes prepared by using calcium phosphate ceramic core coated with various disaccharides for drugs such as insulin and serratiopeptidase through parenteral and oral route respectively^[148-150]. BSA loaded Aquasomes have reported for successful formulation of meter dose inhalers and dry powder inhalers ^[152].

EMULSOMES:

Emulsomes are proven effective delivery of some protein and peptide drugs as shown in Table 12. Emulsomes is a novel vesicular system containing lipodial core surrounded by phospholipid bilayer. The drug is loaded followed by sonication to produce Emulsomes of small size^[153]. Emulsomes with their positive chargewere expected to protect the system from lysosomal degradation and ensure the internalization of the drug^[154,155].

 

 

Table 12: Applications of emulsomes

S. No.	Drug loaded/integrated	Category	Therapeutic application	References
1	Azidothymidine palmitate (AZT-P)	Viral Infections	Development of a drug formulation against HIV	[156,157]
2	Azidothymidine (AZT)	Treatment of intracellular liver infections	----	[158]
3	Amphotericin B (AmB)	Fungal Infections	Treatment of systemic infections with Candida albicans	[159]
			Macrophage-targeted delivery of A m B against visceral leishmaniasis (VL)	[160]
			Treatment of fungal (Aspergillu) infections in the lung	[161]
4	Dithranol	Dermal Therapy	Treatment of psoriasis	[162]
5	Methotrexate (MTX)	Cancer therapy	Treatment of sarcoma of intestinal lymphatic region	[163]
6	Curcumin	Delivery of curcumin into the human liver carcinoma HepG2 cells in vitro	----	[164]
7	Mucosal Vaccine	Autoimmunity	Enhanced immunogenicity	[165]
8	Anti-CD3 monoclonal antibody (mAb)		Enhanced Th2 responses	[166]
9	Insulin	Anti- diabetic	Oral delivery	[166]

 

COMBINATION STRATEGIES:

Combination of two or more strategies for the delivery of proteins and peptide drugs are proven to be effective. These formulation approaches help to overcome the combined problems such as stability, permeability, pre systemic metabolism etc. Edragit L 100 microspheres containing protease inhibitors (aprotinin), Bowman-Birk inhibitors (BBI), Chymostatin (CS), and Trypsin inhibitor (TI) was reported for the delivery of insulin for protection from trypsinic and/or chymotrypsinic degradation^[167].

 

 

 

VITAMIN B₁₂ COATED NANOPARTICLES:

Vitamin B₁₂ transport system has limited capacity. VB₁₂ linkage is itself does not offer protection against proteolysis in the stomach and small intestine. An approach to counter these problems is to employ nanoparticle as carrier for drug. This could amplify the uptake system 10,000 to 100000000 fold and the drug moiety is protected in gastrointestinal milieus. Another benefit could be that the peptide/protein moiety to be delivered need not be linked to the VB₁₂ directly. This would potential benefit in case of peptide molecule like Hirudine and Vasopressin^[167].

 

MUCOADHESIVE/ BIOADHESIVE:

The term bioadhesion can be defined as the state in which two materials, at least one biological in nature, are held together for an extended period of time by interfacial forces^[168]. Adhesion of a matter to this layer (mucus) is called mucoadhesion^[169].

 

The most common approach for the encapsulation of oral insulin is using mucoadhesive polymers, such as chitosan^[170], poly [lactic-co-glycolic acid] (PLGA)^[171], Thiolated polymer and alginate, which have been studied extensively^[172]. Chitosan is a natural non-toxic, biocompatible and biodegradable polymer^[173]. This resulted in the healing of the oral mucosa by arresting epithelial cell division and thus destruction of the cells from the effects of anticancer therapy^[174]. Currently, only two peptide and protein-based drugs Interferon-α and human growth hormone (hGH) that can be given orally are known to be in clinical development^[175].

 

Table 13: Protein and peptide drugsreported to deliver through various mucosal routes

Delivery routes	Protein
Buccal	Insulin, Oxytocin, Vasopressin Analogs, Protirelin and Octreotide multiple polymers
Nasal	Luteinizing-hormone-releasing hormone (LHRH), Thyrotropin-releasing hormone (TRH), Vasopressin, Calcitonin, Oxytocin, ACTH, Glucagon, Insulin, Interferons and Enkephalins
Ocular	Enkephalins, Thyrotropin releasing hormone, LHRH, Glucagon, and Insulin
Rectal	Insulin, Lysozyme, Calcitonin, Gastrin, Pentagastrin
Vaginal	Bromocriptine, Oxytocin, Misoprostol, Calcitonin, LHRH agonists, Human growth hormone And Insulin

 

The absorptive mucosa that has been investigated for delivery of peptides and proteins include buccal, nasal, pulmonary, rectal and vaginal. Nasal delivery has been the most successful–drugs like Buserelin, Desmopressin, Oxytocin, and calcitonin have been available commercially (Table 13). It also helps in delivery of drugs like enkephalin, calcitonin and insulin through   transmucosal membrane^[176].

 

NASAL SPRAYS:

Nasal spray help in the systemic delivery of protein/peptide drugs through nasal route as it has a considerably large absorption area (150cm²) which is highly vascularised and has permeability similar to or higher than the small intestinal mucosa^[177]. Both solution and suspension formulations can be formulated into nasal sprays. Due to the availability of metered dose pumps and actuators, a nasal spray can deliver an exact dose from 25 to 200μm. The particles size and morphology (for suspensions) of the drug and viscosity of the formulation determine the choice of pump and actuator assembly ^[178].

 

A nasal delivery has been studied extensively and is most successful-nasal sprays for Buserelin, Desmopressin, Oxytocin, and calcitonins are already available commercially for delivery of protein and peptides ^[179]. (Table 14)

 

Table 14: Some of the commercially available Nasal sprays^[178-181]:

Drugs	Formulation	Commercial name	Application
Desmopressin acetate	Solution, Spray	Minirin®	Antidiuretic hormones
Salmon calcitonin	Solution, Spray	Miacalcin®	Postmenopausal osteoporosis and Paget’s disease of bones.
Buserelin acetate	Solution, Spray	Suprefact®	Endometriosis
Nafarelin acetate	Solution, Spray	Synarel®	Endometriosis
Oxytocin	Solution, Spray	Syntocinon®	Psychiatric effects
Cyanocobalamine	Solution, Spray	Nascobal®	Allergies
Adrenal corticosteroids	Nasal spray,	Aristocort®	Inflammation
Gentamycin	Nasal spray,	Garamycin®	Antibiotics
Ipratropium bromide	Nasal spray	Atrovent®	Anti cholinergic
LHRH	Nasal spray	Relisorm®	Luteinizing hormones
Insulin	Metered pump sprayer, Metered dose aerosolized spray, Fixed volume aerosol spray, Nasal spray	Exubera®	Antidiabetic
Lypressin	Nasal spray	diapid®	Antidiuretic
Isosorbide dinitrate	Nasal spray	Isordil®	Antianginal drug

 

The peptide is an entirely new approach to treat depression, which has previously relied on medications that primarily block serotonin or norepinephrine transporters. Hence nasal spray also helps in the treatment of depression^[182].

 

Pharmaceutical drugs as well as endogenous hormones such as luteinizing-hormone-releasing hormone LHRH, thyrotropin-releasing hormone (TRH) ^[183,184], vasopressin ^[185], calcitonin, Oxytocin^[186], ACTH^[187], glucagon, insulin^[188,189],interferon ^[190]_, and enkephalin^[191], have been shown to be absorbed nasally in animal and human by nasal sprays. The studies of the nasal delivery of a number of peptide-based pharmaceuticals demonstrated that systemic bioavailability can be improved by nasal route. Morimoto et al. have improved that nasal bioavailability of Calcitonin and Insulin by means of formulation employing Carbopol 941 (corboxy polymethylene, polymer of acrylic acid cross linked with allyl sucrose) and carboxymethyl cellulose (CMC)^[190].

 

Some of the examples of oligopeptides are: 1‐tyrosyl‐1‐tyrosine and its methyl esters, 1‐gycyl‐1‐tyrosine,1‐glycyl‐1‐tyrosinamide (Dipeptides), Thyrotropin‐releasing hormone (TRH) (Tripeptides), Leucin‐enkephalin, met‐enkephamide (Pentapeptides) and for polypeptides are Vasopressin, Oxytocin (Nonapeptides), LHRH (Decapeptides), Substance P (Undecapeptides), Glucagon, Calcitonin and Adrenal corticotropic hormone (ACTH).

 

CONCLUSION:

Therapeutic proteins and peptides are gaining importance in healthcare system. In recent years, numerous therapeutically potent protein and peptide drugs have been developed. Although highly potent, one of the major challenges to the successful clinical use of this therapeutics is lack of an effective delivery method. Parenteral delivery remains still upfront even though abundant efforts have been put toward delivering protein and peptide via noninvasive routes. Hence, there is large scope to develop viable delivery systems for the efficient use of these complex therapeutic agents in biologically active form.

 

REFERENCE:

1.        Tan ML, Chong PF, Dass CR , Recent developments in liposomes, Microparticles and nanoparticle for protein and peptide drug delivery. Peptides. 2010 Jan; 31(1):184-93. Doi: 10.1016/j.peptides.2009.10.002. Epub 2009 Oct 9

2.        Patel A, Cholkar K, Mitra AK. Recent Developments in Protein and Peptide Parenteral Delivery Approaches. There Delivery. 2014;5(3):337–652

3.        V Rajesh Babu, Syeda Rana Nikhat, P Nivethithai, SH Areefulla; Approaches and Challenges of Protein and Peptide Drug Delivery Systems; Research Journal of Pharmacy and Technology,0974-360X,Pages :  379-384 ,Vol No : 3, , 2010.

4.        Https://labels.fda.gov/

5.        Pandey Rishabh, Singh AV, Pandey Awanish, Tripathi Poonam, SK Majumdar, Nath LK. Journal; Protein and Peptide Drugs: A Brief Review; Research Journal of Pharmacy and Technology; 0974-360X; Pages :  228-233,Volume No: 2 , 2009   

6.        M. Sunitha Reddy, Sama Mallikarjun Reddy, A. Mahesh Chandra, B. Sai Santhosh, Ch. Surekha, K. Naveen; Novel Approaches for Delivery of Proteins and Peptides – A Review; Research Journal of Pharmaceutical Dosage Forms and Technology; 0975-4377; Pages :  7-11,vol no:5,2013.

7.        Genetic Engineering and Biotechnology News. Top 20 Best-Selling Drugs of 2012.www.genengnews.com/insight-and-intelligence/top-20-best-selling-drugs-of-2012/77899775

8.        Department of Health and Human Services. US FDA. Background document for meeting of advisory   committee for reproductive health drugs and drug safety and risk management advisory committee. Vol. 113. MD, USA: 2013

9.        Mitchell, M. The medicines company reports full year and fourth quarter 2011 financial results. NJ, USA: 2012

10.      Department of Health and Human Services. US FDA; Gill R. Desmopressin acetate drug use review in paediatric population. 2010

11.      Shimanovich, U. Et al. Protein micro- and nano-capsules for biomedical applications. Chem. Soc. Rev. 43, 1361–1371;2014

12.      Ashok Kumar, O.P. Yadav; Equilibrium Dialysis Studies on the Interaction of some Surfactants with Poly(N-vinyl-2-pyrrolidone) and Bovine Serum albumin; Asian Journal of Research in Chemistry;0974-4150, Pages: 552-555, Volume No.: 5, 2012.

13.      Martins, M. Et al.; In vitro and computational studies of transdermal perfusion of nanoformulations containing a large molecular weight protein. Colloids Surf. B Biointerfaces 108, 271–278,2013

14.      Nitta, S.K. and Numata, K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int. J. Mol. Sci. 14, 1629–1654;2013

15.      Lohcharoenkal, W. Et al.; Protein nanoparticles as drug delivery carriers for cancer therapy. Biomed. Res. Int. 2014, 180549;2014

16.      Tahara, Y. Et al.; Solid-in-oil dispersion: a novel core technology for drug delivery systems. Int. J. Pharm. 438, 249–257.2012

17.      Jun, J.Y. et al. Preparation of size-controlled bovine serum albumin (BSA) nanoparticles by a modified desolvation method. Food Chem. 127, 1892–1898;2011

18.      Tesch, S. Et al. Production of emulsions in high-pressure homogenizers – part I: disruption and stabilization of droplets. Chem. Eng. Technol. 26, 569–573;2003

19.      Wang et al., 2010Wang T, Wang N, Hao A, et al.; Lyophilization of water-in-oil emulsions to prepare phospholipid-based anhydrous reverse micelles for oral peptide delivery. Eur J Pharm Biopharm 39:373–9;2010.

20.      Liu, Dongyun, Kobayashi, Taku, Russo, In vitro and in vivo evaluation of a water-in-oil microemulsion system for enhanced peptide intestinal delivery, AAPS J. 15(1), 288-298,2013.

21.      Jadupati Malakar, Suma Oomen Sen, Amit Kumar Nayak, Development and evaluation of microemulsions for transdermal delivery of insulin, ISRN Pharma., Article ID 780150, 1-7,2011.

22.      Y. Arribat, Y. Talmat-Amar, A. Systemic delivery of P42 peptide: a new weapon to fight huntington’s disease, Acta. Neuropath. Comm. 2(86),1- 17, 2014.

23.      Wei Wang, Manmohan Singh, Selection of adjuvants for enhanced vaccine potency, World J. Vacc., 1, 33-78, 2011.

24.      Ahmet Dogrul, Seyda Akkus Arslan, and Figen Tirnaksiz. Water/oil type microemulsion systems containing lidocaine hydrochloride: in vitro and in vivo evaluation, J. Microencap, 1–13,2014.

25.      Macedo, Ludmila Branco, Evaluation of different adjuvants formulations for bluetongue vaccine, Braz. Arch. Biol. Technol. 56(6), 932-941,2013.

26.      Derya Ilem-Ozdemir, Neslihan Ustundag-Okur. Effect of microemulsion formulation on biodistribution of 99mtc-aprotinin in acute pancreatitis models induced rats, Drug Deliv., 23(8), 3055-3062,2016.

27.      H. Rachmawati, B.M. Haryadi, K. Anggadiredja, Intraoral film containing insulin-phospholipid microemulsion: formulation and in vivo hypoglycemic activity study, AAPS Pharmscitech., 16(3), 692-703, 2015.

28.      Fujii S, Yokoyama T, Ikegaya K, Sato F, Yokoo NJ; Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs. Pharm Pharmacol; 37(8):545-9,Aug 1985.

29.      Bernkop SA. The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins; J Control Release; 52:1–16;1998.

30.      Hoang VD, Uchenna AR, Mark J, Renaat K, Norbert V. Characterization of human nasal primary culture systems to investigate peptide metabolism. Int. J. Pharm; 238: 247-25;2002.

31.      O' Hagan DT, Illum L. Absorption of peptides and proteins from the respiratory tract and the potential for development of locally administered vaccine. Crit Rev; There Drug Carrier Systm; 7:35–97;1990

32.      P. Muralidhar, S. Babajan, E. Bhargav, C. Sowmya, Protien and Peptide Based Drug Delivery 17-01-2017

33.      Fujii S, Yokoyama T, Ikegaya K, Sato F, Yokoo NJ Pharm Pharmacol. Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs; 37(8):545-9. Aug 1985

34.      Ziv E, Lior O, Kidron M, Absorption of protein via the intestinal wall. A quantitative model. Biochem Pharmacol.; 36(7):1035-9;1Apr 1987

35.      Uchiyama T, Kotani A, Kishida T, et al. Effects of various protease inhibitors on the stability and permeability of [d-Ala2, d-Leu5] enkephalin in the rat intestine: comparison with leucine enkephalin. J. Pharm. Sci ;87(4):448–452;1998.

36.      Mcdonald JK, Reilly TJ, Zeitman BB, Ellis S. Dipeptidyl arylamidase II of the pituitary. Properties of lysylalanyl-beta-naphthylamide hydrolysis: inhibition by cations, distribution in tissues, and subcellular localization. J. Biol. Chem;243(8):2028–2037;1968.

37.      The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins. Bernkop-Schnürch A, J Control Release; 52(1-2):1-16;2Mar 1998.

38.      Bernkop-Schnurch A, Marschutz MK. Development and in vitroevaluation of systems to protect peptide drugs from aminopeptidase. N. Pharm. Res;14(2):181–185;1997.

39.      Bacitracin, its manufacture and uses.Hickey RJProg Ind Microbiol; 5():93-150;1964.

40.      SM Sivakumar, L Nirmala, R Swarnalakshmi, L Sivakumar, B Anilbabu, TS Shanmugarajan, J Anbu, V Ravichandran, N Sukumaran; Preparation and In vitro Evaluation of Hepatitis B Vaccine Encapsulated Chitosan Microparticles; Research Journal of Pharmacy and Technology; 0974-360X; vol no: 2, Pages: 117-119,2009.

41.      Viviansaez,,Joséramón Hernández, Carlos Peniche , microspheres as delivery system for the control release of proteinsand peptides, Biotecnología Aplicada;24:108-116;2007.

42.      Thanou M, Verhoef JC, Junginger HE. Chitosan and its derivatives as intestinal absorption enhancers. Adv. Drug Deliv.; 50(Suppl. 1): S91–S101; Rev. 2001

43.      Cano-Cebrian M, Zornoza T, Granero L, Polache A. Intestinal absorption enhancement via the paracellular route by fatty acids, chitosans, and others: a target for drug delivery. Curr. Drug Delivery; 2(1):9–22;2005. [pubmed: 16305404]

44.      Sayani AP, Chien YW systemic delivery of peptides and proteins across absorptive mucosea, 01 Jan 1996

45.      Morishita M, Kamei N, Ehara J, Isowa K, Takayama K. A novel approach using functional peptides for efficient intestinal absorption of insulin. J. Control. Release.; 118(2):177–184;2007. [pubmed: 17270307]

46.      N.N. Salama, N.D. Eddington, A. Fasanotight junction modulation and its relationship to drug delivery Adv. Drug Delivery Rev., 58 (2); pg: 15-28;2006.

47.      C. Prego, M. Garcia, D. Torres, M.J. alonsotransmucosal Macromolecular Drug Delivery J. Controlled Release, pg. 151-162;2005.

48.      A. Bernkop-schnurchthiomers: a new generation of mucoadhesive polymers Adv. Drug Del, pg:1569-1582, Rev., 57 (11) 2005.

49.      A. Fasano, S. Uzzaumodulation of intestinal tight Junctions zona occludens toxin permits enteral administration of insulin and other macromolecules in an animal model J. Clin. Invest., pp. 1158-1164; rev:99 (6) 1997.

50.      Herwadkar A, Banga AK. Peptide and protein transdermal drug delivery. Drug Discov. Today Technol;9(2): e147–e154;. 2012

51.      Sibalis D., Transdermal drug applicator. U. S. Patent 4,708,716, 1987.

52.      Meyer B. R., Electro‐osmotic transdermal drug delivery, in:  1987 Conference Proceedings on the Latest Developments in Drug Delivery Systems, Aster Publishing, Eugene, Oregon, 40,1987.

53.      Meyer et al. Transdermal delivery of human insulin to albino rabbits using electrical current. Am. J. Med. Sci, 297:321‐325, 1989.

54.      Chein Y. W., Siddiqui O. And Liu J. C., Transdermal iontophoretic delivery of therapeutic peptides/proteins. I. Insulin. Ann. N. Y. Acad. Sci., 507:32‐51,1988.

55.      Pandey Rishabh, 2Singh AV, Pandey Awanish, Tripathi Poonam, SK Majumdar, Nath LK. Journal; Protein and Peptide Drugs: A Brief Review; Research Journal of Pharmacy and Technology;0974-360X; Volume No: 2 Pages:  228-233, 2009.  

56.      Wilson, EJ. Three Generations: The Past, Present, and Future of Transdermal Drug Delivery, Systems. Pharmcon; SC, USA: 2011.

57.      H. Chung, J. Kim, J.Y. Um, I.C. Kwon, S.Y. Jeongself-Assembled ‘Nanocubicle’ As a Carrier for Peroral Insulin delivery diabetologia, pg. 448-451;45 (3) (2002).

58.      Mir S Adil, S. Muzammil Hassan, Azizullah Ghouri, M. Nematullah K., M. Amer K., Ihtisham S; Oral Insulin: To Make Needles Needless; Research Journal of Pharmaceutical Dosage Forms and Technology; 0975-4377; Volume No. : 6 , Pages :58-61 , 2014 .

59.      H.Y. Sang, T.G. park biodegradable nanoparticles containing protein, fatty acid complexes for oral delivery of salmon calcitonin J. Pharm. Sci;pg. 488-495; 93 (2) 2004.

60.      A.C. Foss, T. Goto, M. Morishita, N.A. Peppas Development of acrylic-based copolymers for oral insulin delivery; Eur. J. Pharm. Biopharm.;pg. 163-169;57 (10) 2004.

61.      P.P. constantinides; Lipid microemulsions for improving drug dissolution and oral absorption; physical and biopharmaceutical aspects Pharm. Res.; 12(11):1561-72. Nov 1995.

62.      T. Mizuma, K. Ohta, A. Koyanagi, S. Awazu. Improvement of intestinal absorption of leucine enkephalin by sugar coupling and peptidase inhibitors J. Pharm. Sci., pg. 854-857;85 (8) 1996.

63.      Y. Pan, Y.J. Li, H.Y. Zhao, J.M. Zheng, H. Xu, G. Wei, et al. Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo Int. J. Pharm.;  pg. 139-147;249 (1–2) 2002

64.      C. Leroux, R. Cozens, J.L. Roesel, B. Galli, E. Doelker, R. Gurnyph-Sensitive nanoparticles an effective means to improve the oral delivery of HIV-1 protease inhibitors in dogs; Pharm. Res., 13 (3) ;pg. 485-487;1996.

65.      H. Yoshida, C.M. Lehr, W. Kok, H.E. Junginger, J.C. Verhoef, J.A. bouwistraniosomes for oral delivery of peptide drugs J. Controlled Release, 21 (1–3); pg. 145-153;1992.

66.      Ducat E, Deprez J, Gillet A, et al. Nuclear delivery of a therapeutic peptide by long circulating ph-sensitive liposomes: benefits over classical vesicles. Int. J. Pharm.; 420(2):319–332;2011. [pubmed: 21889584]

67.      Sonal Gupta, Arushi Jain, Mainak Chakraborty, Jasjeet K Sahni, Javed Ali and Swetha Dang, oral delivery of therapeutic proteins and peptides, 20 july 2013

68.      Gabizon A, Martin F. Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Rationale for use in solid tumours. Drugs. 54(Suppl. 4):15–21;1997. [pubmed: 9361957]

69.      Frkanec R, Noethig-Laslo V, Vranesic B, et al. A spin labelling study of immunomodulating peptidoglycan monomer and adamantyl tripeptides entrapped into liposomes. Biochimica et Biophysica Acta (BBA) – Biomembranes.; 1611:187–96;2003.

70.      Brgles M, Mirosavljevic K, Noethig-Laslo V, et al. Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies. Int J Biol Macromol;40(4):312-8;10 Mar2007.

71.      Plum SM, Holaday JW, Ruiz A, et al. Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor development. Vaccine.; 19:1294–303;2000.

72.      Luo X, Belcastro R, Cabacungan J, et al. Transfection of lung cells in vitro and in vivo: effect of antioxidants and intraliposomal bfgf. Am J Physiol – Lung C.; 284: L817–L25; 2003.

73.      Dai C, Wang B, Zhao H, et al. Factors affecting protein release from microcapsule prepared by liposome in alginate. Colloid Surface B.; 42:253–8;2005.

74.      Murakami S, Ono T, Sakai S, et al. Effect of Diglucosamine on the Entrapment of Protein into Liposomes. J Lipos Res.; 16:103–12.2006.

75.      Takeuchi H, Matsui Y, Yamamoto H, et al. Mucoadhesive properties of carbopol or chitosan-coated liposomes and their effectiveness in the oral administration of calcitonin to rats. J Control Release.;86:235–42;2003.

76.      Thongborisute J, Tsuruta A, Kawabata Y, et al. The effect of particle structure of chitosan-coated liposomes and type of chitosan on oral delivery of calcitonin. J Drug Targ. 2006; 14:147–54.

77.      Ye Q, Asherman J, Stevenson M, et al. Depofoam(TM) technology: a vehicle for controlled delivery of protein and peptide drugs. J Control Release.;64:155–66;2000.

78.      Kullberg E, Wei Q, Capala J, et al. EGF-receptor targeted liposomes with boronated acridine: Growth inhibition of cultured glioma cells after neutron irradiation. Int J Radiat Biol.; 81:621–9;2005.

79.      Gorodetsky R, Levdansky L, Vexler A, et al. Liposome transduction into cells enhanced by haptotactic peptides (Haptides) homologous to fibrinogen C-termini. J Control Release.;95:477–88;2004.

80.      Arifin DR, Palmer AF; Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering. Biotechnol Prog.; 19:1798–811;2003.

81.      Patton JN, Palmer AF; Photopolymerization of bovine hemoglobin entrapped nanoscale hydrogel particles within liposomal reactors for use as an artificial blood substitute. Biomacromolecules; 6:414–24;2005.

82.      Teiji O, Tetsuhiro K, Yoshitaka O, et al. Hemodilution with liposome-encapsulated low-oxygen-affinity hemoglobin does not attenuate hypothermic cerebral ischemia in rats. Journal of Artificial Organs; 8:263–9;2005.

83.      Xi J, Guo R. Interactions between flavonoids and hemoglobin in lecithin liposomes. Int J Biol Macromol.;40(4):305-11; 10 Mar 2007.

84.      Visser CC, Stevanovic S, Voorwinden LH, et al. Targeting liposomes with protein drugs to the blood-brain barrier in vitro. Eur J Pharm Sci.; 25:299–305;2005.

85.      Visser CC, Stevanovic S, Voorwinden LH, et al. Targeting liposomes with protein drugs to the blood-brain barrier in vitro. Eur J Pharm Sci; 25:299–305;2005.

86.      Zhang N, Ping QN, Huang GH, et al. Investigation of lectin-modified insulin liposomes as carriers for oral administration. Int J Pharm.; 294:247–59;2005.

87.      Goto T, Morishita M, Nishimura K, et al. Novel mucosal insulin delivery systems based on fusogenic liposomes. Pharm Res; 23:384–91;2006.

88.      Badiee A, Jaafari MR, Khamesipour A. Leishmania major: Immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes. Exp Parasitol.; 115:127–34;2007.

89.      Langston MV, Ramprasad MP, Kararli TT, et al. Modulation of the sustained delivery of myelopoietin (Leridistim) encapsulated in multivesicular liposomes (depofoam) J Control Release.;89:87–99;2003.

90.      Guo J, Ping Q, Jiang G, et al. Chitosan-coated liposomes: characterization and interaction with leuprolide. Int J Pharm; 260:167–73;2003.

91.      Arulsudar N, Subramanian N, Mishra P, Chuttani K, Sharma RK, Murthy RS; Preparation, characterization, and biodistribution study of technetium-99m -labeled leuprolide acetate-loaded liposomes in Ehrlich ascites tumor-bearing mice.;AAPS pharmsci.; 6(1):E5;6Feb2004.

92.      Shahiwala A, Misra A; A preliminary pharmacokinetic study of liposomal leuprolide dry powder inhaler: a technical note. AAPS pharmscitech.; 6: E482–6;2005.

93.      Carafa M, Marianecci C, Annibaldi V, et al;Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: Development, characterization and interaction with leuprolide. Int J Pharm.; 325:155–62;2006.

94.      Xie Y, Ye L, Zhang X, et al;Transport of nerve growth factor encapsulated into liposomes across the blood-brain barrier: In vitro and in vivo studies; J Control Release;105:106–19;2005.

95.      Ye Q, Asherman J, Stevenson M, et al. Depofoam(TM) technology: a vehicle for controlled delivery of protein and peptide drugs. J Control Release; 64:155–66;2000.

96.      Ramprasad MP, Amini A, Kararli T, et al; The sustained granulopoietic effect of progenipoietin encapsulated in multivesicular liposomes. Int J Pharm;261(1-2):93-103; 11 Aug 2003.

97.      Amselem S, Domb AJ, Alving CR. Lipospheres as a vaccine carrier system: effects of size, charge, and phospholipid composition. Vaccine.;1:383–95 2003;261:93–103,1992.

98.      Ruzica GR, Karmela B, Zeljka P, et al. High efficiency entrapment of superoxide dismutase into mucoadhesive chitosan-coated liposomes. Eur J Pharm Sci.; 15:441–8;2002.

99.      Torchilin VP, Rammohan R, Weissig V, Levchenko TS; AT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors;Proc Natl Acad Sci U S A; 98(15):8786-91;17Jul2001

100.   Mashaghi, S.; Jadidi, T.; Koenderink, G.; Mashaghi, A. Lipid Nanotechnology. Int. J. Mol. Sci., 14, 4242-4282;2013.

101.   Müller, R.H.; Mäder, K.; Gohla S;Solid lipid nanoparticles (SLN) for controlled drug delivery–A review of the state of the art. Eur. J. Pharm. Biopharm., 50, 161–177;2000.

102.   Antonio j.almedia Eliana southo; solid lipid nano particles as a drug delivery system for proteins and peptides Advanced drug delivery reviews ;volume 59,issue 6,pages 478-490,july 2007.

103.    Zur Mühlen, A.; Schwarz, C.; Mehnert, W. Solid lipid nanoparticles (SLN) for controlled drug delivery–Drug release and release mechanism. Eur. J. Pharm. Biopharm., 45, 149–155;1998.

104.   AL-Haj, N.A.; Abdullah, R.; Ibrahim, S.; Bustamam, A. Tamoxifen drug loading solid lipid nanoparticles prepared by hot high pressure homogenization techniques. Am. J. Pharmacol.Toxicol., 3, 219–224;2008.

105.   Harivardhan Reddy, L.; Vivek, K.; Bakshi, N.; Murthy, R.S.R. Tamoxifen citrate loaded solid lipid nanoparticles (SLN™): Preparation, characterization, in vitro drug release, and pharmacokinetic evaluation. Pharm. Dev. Technol, 11, 167–177;2006.

106.   Gualbert, J.; Shahgaldian, P.; Coleman, A.W. Interactions of amphiphilic calyx[4]arene-based ;solid lipid nanoparticles with bovine serum albumin. Int. J. Pharm., 257, 69–73;2003.

107.   Olbrich, C.; Geßner, A.; Kayser, O.; Müller, R.H. Lipid-drug conjugate (LDC) nanoparticles asnovel carrier system for the hydrophilic antitrypanosomal drug diminazenediaceturate. J. Drug Target., 10, 387–396;2002.

108.   Müller, R.H.; Runge, S.A.; Ravelli, V.; Thünemann, A.F.; Mehnert, W.; Souto, E.B.; Cyclosporine-loaded solid lipid nanoparticles (SLN®): Drug-lipid physicochemical interactions and characterization of drug incorporation. Eur. J. Pharm. Biopharm., 68, 535–544;2008.

109.   Penkler, L.; Müller, R.H.; Runge, S.; Ravelli, V. Pharmaceutical cyclosporin formulation with improved biopharmaceutical properties, improved physical quality and greater stability, and method for producing said formulation. US Pat. No. 6,551,619, 2003.

110.   Ugazio, E.; Cavalli, R.; Gasco, M.R. Incorporation of cyclosporine A in solid lipid nanoparticles (SLN). Int. J. Pharm., 24, 341–344,2002.

111.   Bajoria, R.; Sooranna, S.R. Liposome as a drug carrier system: Prospects for safer prescribing during pregnancy: A review. Placenta, 19, 265–287,1998.

112.   García-Fuentes, M.; Torres, D.; Alonso, M.J. Design of lipid nanoparticles for the oral delivery of hydrophilic macromolecules. Colloids Surf. B Biointerf., 27, 159–168,2002.

113.   Zhang, N.; Ping, Q.; Huang, G.; Xu, W.; Cheng, Y.; Han, X. Lectin-modified solid lipid   nanoparticles as carriers for oral administration of insulin. Int. J. Pharm., 327, 153–159;2006.

114.   Caliceti, P.; Brossa, A.; Salmaso, S.; Bersani, S.; Elvassore, N.; Bertucco, A. Preparation of protein loaded solid lipid nano-particles by compressed fluid process. Proc. Int. Symp. Control. Rel. Bioact. Mater., 23, 383,2006.

115.   Cavalli, R.; Bocca, C.; Miglietta, A.; Caputo, O.; Gasco, M.R. Albumin adsorption on stealth and non-stealth solid lipid nanoparticles. STP Pharma. Sci., 9, 183–189,1999.

116.   Morel, S.; Gasco, M.R.; Cavalli, R. Incorporation in lipospheres of [D-Trp-6] LHRH. Int. J. Pharm., 105, R1–R3,1994.

117.   Almeida, A.J.; Runge, S.; Müller, R.H. Peptide-loaded solid lipid nanoparticles (SLN): Influence of production parameters. Int. J. Pharm., 149, 255–265;1997.

118.   Videira, M.; Azevedo, A.F.; Almeida, A.J. Entrapment of a high molecular weight protein intosolid lipid nanoparticles. Proc. 2nd World Meeting APV/APGI, Paris; pg. 629–630; May 1998.

119.   Morel, S.; Ugazio, E.; Cavalli, R.; Gasco, M.R. Thymopentin in solid lipid nanoparticles. Int. J.Pharm., 132, 259–261,1996.

120.   Vauthier, C.; Bouchemal, K. Methods for the preparation and manufacture of polymeria nanoparticles. Pharm. Res., 26, 1025–1058;2009

121.   Chen, F.; Zhang, Z.-R.; Yuan, F.; Qin, X.; Wang, M.; Huang, Y. In vitro and in vivo study of Ntrimethylchitosan nanoparticles for oral protein delivery. Int. J. Pharm., 349, 226–233;2008.

122.   Chan JM, Valencia PM, Zhang L, et al. Polymeric nanoparticles for drug delivery. Methods Mol Biol 624:163–75;2010.

123.   Sarmento, B.; Ribeiro, A.; Veiga, F.; Sampaio, P.; Neufeld, R.; Ferreira, D. Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharm. Res., 24, 2198–2206;2007.

124.   Tan ML, Choong PF, Dass CR (2009) Cancer, chitosan nanoparticles and catalytic nucleic acids. J Pharm Pharmacol 61: 3-12.;. doi: 10.1211/jpp/61.01.0002. Jan 2009

125.   Vauthier, C.; Bouchemal, K. Methods for the preparation and manufacture of polymeric nanoparticles. Pharm. Res., 26, 1025–1058; 2009

126.   Christine Vauthier and Kawthar Bouchemal;,Methods for the Preparation and Manufacture of Polymeric Nanoparticles, Pharmaceutical Research, 1027, 26, 2009.

127.   Xuan B, mcclellan DA, Moore R, Chiou GC. Alternative delivery of insulin via eye drops. Diabetes Technol Ther;7(5):695–8;2005.

128.   Ahsan F, Arnold J, Meezan E, Pillion DJ. Enhanced bioavailability of calcitonin formulated with alkylglycosides following nasal and ocular administration in rats. Pharm Res. ;18(12):1742–6;2001.

129.   Chiou GC, Shen ZF, Zheng YQ, Li BH. Enhancement of systemic delivery of met-enkephalin and leu-enkephalin eyedrops with permeation enhancers. Methods Find Exp Clin Pharmacol.;14(5):361–6;1992.

130.   Orange book: approved drug products with therapeutic equivalence evaluations. http://www.accessdata.fda.gov/scripts/cder/ob/default.cfm.

131.   Ch. Prabhakar, K. Bala Krishna ;A Review on Polymeric Nanoparticles; Research Journal of Pharmacy and Technology ;0974-360X;Volume No :4 , Pages :  496-498 ,2011; 

132.   Oliyai R, Stella VJ. Prodrugs of peptides and protein for improved formulation and delivery. Annu. Rev. Pharmcol. Toxicol.; 32: 521-544;1993.

133.   Miyamoto S, Takaoka K, Okada T, Yoshikawa H, Hashimoto J, Suzuki S, Ono K. Polylactide acid –polyethylene glycol block copolymer: a new biodegradable synthetic carrier for bone morphogenetic protein. Clin. Orthopaedics Related Res.; 294: 333-343;1993.

134.   Gombotz WR, Pankey SC, Bouchard LS, Ranchalis J, Puolakkainen P. Controlled release of TGF-beta (1) from a biodegradable matrix for bone regeneration. J. Biomater. Sci. Polymer; 5: 49-63;1993.

135.   Heya, T, Okada H, Ogawa Y, Toguchi H. Factors influencing the profiles of TRH release from copoly (d,l-lactic/glycolic acid) microspheres. Int. J. Pharm.; 72: 199-205;1991.

136.   Mariette Β, Coudane J, Vert M, Gautier JC, Moneton P. Release of the GRF29NH2 analog of human GRF44NH2 from a PLA/ GA matrix. J Contrl. Rel.; 237-246,1993.

137.   Bodmer D, Kissel T, Traechslin E. Factors influencing the release of peptides and proteins from biodegradable parenteral depot systems. J. Contrl. Rel.; 21: 129-138;1992.

138.   Yamakawa I, Tsushima Y, Machida R, Watanabe S. Preparation of neurotensin analogue-containing poly (dl-lactic acid) microspheres formed by oil in water solvent evaporation. J. Pharm Sci.; 81: 899-903;1992.

139.   Sanchez Α, Vila-Jato JL, Alonso MJ. Development of biodegradable microspheres and nanospheres for the controlled release of cyclosporine A. Int. J. Pharm.; 99(2-3): 263-273;1993.

140.   Reid RH, Boedeker EC, Mcqueen CE, Davis D, Tseng LY, Kodak J, Sau K. Preclinical evaluation of microencapsulated CFA/II oral vaccine against enterotoxi- genic E. Coli. Vaccine; 11(2): 159-167;1993.

141.   Almeidia AJ, Alpar HO, Williamson D, Brown MRW. Poly (lactic acid) microspheres as immunological adjuvants for orally delivered cholera toxin B subunit. 643rd Meeting of the Biochemical Society (Warwick, UK). Biochem. Soc.Trans.; 20: 316S;1992.

142.   Singh M, Sing, O, Singh Α, Talwar GP. Immunogenicity studies on diphtheria toxoid loaded biodegradable microspheres. Int. J. Pharm.; 85: R5-R8;1992.

143.   Jeffery H, Davis SS, O’Hagen DT. The preparation and characterization of poly(lactide-co-glycolide) microparticle. II. The entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique. Pharm. Res; 10: 362-368;1993.

144.   Alonso MJ, Cohen S, Park TG, Gupta RK, Siber GR, Langer R. Determinants of release rate of tetanus vaccine from polyester microspheres. Pharm. Res.; 10 (7): 945-953;1993.

145.   Priyanka R. Adhau; A Study to Evaluate the Effectiveness of Demonstration on self Insulin Administration with Regards to Practice among Diabetes Mellitus Clients in Selected Diabetic Clinics in Metropolitan City; International Journal of Nursing Education and Research;DOI:10.5958/2454-2660.2017.00039.4; Volume No. : 5 , Pages :  185-187 ; 2017

146.   Development and in vivo evaluation of a new oral nanoparticulate dosage form for leuprolide based on polyacrylic acid. Iqbal J, Vigl C, Moser G, Gasteiger M, Perera G, Bernkop-Schnürch adrug Deliv.; 18(6):432-40;Aug2011.

147.   Deutel B, Greindl M, Thaurer M, Bernkop-Schnürch A. Novel insulin thiomer nanoparticles: in vivo evaluation of an oral drug delivery system. Biomacromolecules. 9(1):278–85;2008.

148.   N.L Prasanthi, S.S. Manikiran, C. Sowmya Krishna, N. Rama Rao; Aquasomes: Role to Deliver Bioactive Substances; Research Journal of Pharmaceutical Dosage Forms and Technology;0975-4377; Volume No. :2, Pages :356-360, 2010.

149.   Cherian, A. K.; Rana, A. C.; Jain, S. K. Drug Dev Ind Pharm., 26, 459-63,2000.

150.   Rawat, M.; Singh, D.; Saraf. S.; Saraf, S. Drug Dev Ind Pharm., 34, 181-8,2008.

151.   Kim, I.S., Kim, S.H; Development of polymeric nanoparticulate drug delivery system. In vitro characterization of nanoparticles based on sugar containing conjugates. International Journal of Pharmaceutics. 245, 67–73; 2002.

152.   Fadi Abdulrazzaq; Aquasomes as a drug delivery system for proteins and peptides 26 Oct 2016.

153.   Vyas SP, Subhedar R, Jain S. Development and characterization of emulsomes for sustained and targeted delivery of an antiviral agent to liver. J Pharm Pharmacol; 58:321-6;2006.

154.   Mehmet H. Ucisik, Uwe B. Sleytr and Bernhard Schuster; Emulsomes Meet S-layer Proteins: An Emerging Targeted Drug Delivery System; Curr Pharm Biotechnol.; 16(4): 392–405; Apr 2015

155.   Phillips NC, Tawashi R, Heiati H ;Drug retention and stability of solid lipid nanoparticles containing azidothymidine palmitate after autoclaving, storage and lyophilization., J Microencapsul.; 15(2):173-84;Mar-Apr 1998.

156.   Heiati H, Tawashi R, Shivers RR, Phillips NC. Solid lipid nanoparticles as drug carriers. I. Incorporation and retention of the lipophilic prodrug 3'-azido-3'-deoxythymidine palmitate. Int. Pharmaceut. ;146(1 ):123–131; 1997.

157.   Drug retention and stability of solid lipid nanoparticles containing azidothymidine palmitate after autoclaving, storage and lyophilization. Heiati H, Tawashi R, Phillips NCJ Microencapsul; 15(2):173-84. Mar-Apr 1998.

158.   Development and characterization of emulsomes for sustained and targeted delivery of an antiviral agent to liver. Vyas SP, Subhedar R, Jain S, J Pharm Pharmacol.; 58(3):321-6; Mar 2006.

159.   Efficient treatment of murine systemic infection with Candida albicans using amphotericin B incorporated in nanosize range particles (emulsomes). Kretschmar M, Amselem S, Zawoznik E, Mosbach K, Dietz A, Hof H, Nichterlein T, Mycoses.; 44(7-8):281-6;2001.

160.   Development and evaluation of tripalmitin emulsomes for the treatment of experimental visceral leishmaniasis, Pal A, Gupta S, Jaiswal A, Dube A, Vyas SP, J Liposome Res.; 22(1):62-71; Mar 2012.

161.   Functionalized nanocarrier(s) to image and target fungi infected immune cells. Vyas SP, Khatri K, Goyal AK, Med Mycol.; 47 Suppl 1(): S362-8;2009.

162.   Improved therapeutic performance of dithranol against psoriasis employing systematically optimized nanoemulsomes. Raza K, Katare OP, Setia A, Bhatia A, Singh B, J Microencapsul.; 30(3):225-36;2013.

163.   Engineered chylomicron mimicking carrier emulsome for lymph targeted oral delivery of methotrexate. Paliwal R, Paliwal SR, Mishra N, Mehta A, Vyas SP ,Int J Pharm.; 380(1-2):181-8;1 Oct 2009.

164.   Characterization of curcuemulsomes: nanoformulation for enhanced solubility and delivery of curcumin. ,Ucisik MH, Küpcü S, Schuster B, Sleytr UB J Nanobiotechnology.; 11():37;6 dec 2013.

165.   Vancott TC, Kaminski RW, Mascola JR, Kalyanaraman VS, Wassef NM, Alving CR, Ulrich JT, Lowell GH, Birx DL. HIV-1 neutralizing antibodies in the genital and respiratory tracts of mice intranasally immunized with oligomeric gp160. J. Immunol. ;160(4):2000–2012;1998.

166.   Wu HY, Maron R, Tukpah AM, Weiner HL. Mucosal anti-CD3 monoclonal antibody attenuates collagen-induced arthritis that is associated with induction of LAP+ regulatory T cells and is enhanced by administration of an emulsome-based Th2-skewing adjuvant. J. Immunol. ;185(6):3401–3417;2010.

167.   P. Muralidhar, S.Babajan, E.Bhargav, C.Soumya ;protein peptide based drug delivery system,; Int J Pharm, Sci.Rev.Res,42(1), January to February 2017.

168.   Good WR; Transdermal nitro-controlled delivery of nitroglycerin via the transdermal route. Drug Dev Ind Pharm.; 9:647–70;1983.

169.    J.D. Smart. The basics and underlying mechanisms of mucoadhesion. Adv Drug Deliv Rev. 57:1556-1568 ;2005.

170.   E. Mathiowitz, J.S. Jacob, Y.S. Jong, G.P. Carino, D.E. Chickering, P. Chaturvedi. Biologically erodible microspheres as potential oral drug delivery systems ;Nature, 386 (6623) , pg. 410-414;1997.

171.   C. Damge, C. Michael, M. Aprahamian, P.couvreurnew approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as oral carrier  Diabetes, 37 (2) , pg. 247-251;1988.

172.   S. Takka, F. Acarturkcalcium alginate Microparticles for oral administration. I: effect of sodium alginate type on drug release and drug entrapment efficiency J. Microencap., 16 (3), pg. 275-290;1999.

173.   R. Hejazi, M. Amijichitosan-based gastrointestinal delivery system J. Controlled Release, 89 (2) , pg. 151-165;2003

174.   S. Senel, M.J. Kremer, S. Kas, P.W. Wertz, A. A. Hincal, C.A. squierenhancing effect of chitosan on peptide drug delivery across buccal mucosa Biomaterials, 21 (20) , pg. 2067-2071;2000.

175.   J. Renukuntla, A.D. Vadlapudi, A. Patel, S.H.S.Boddu, A.K. mitraapproaches for enhancing oral bioavailability of peptides and proteins Int. J. Pharm., 447 (1–2) , pg. 75-93;2013

176.   Sayaniap,cheinyw  systemic delivery of peptides and proteins across absortive mucosae ,critical reiews in therapeutic drug carrier systems 13 (1-2): 85-184 ;01jan1996

177.   Mo R, Jiang T, Di J, Tai W, Gu Z Emerging micro-and nanotechnology based synthetic approaches for insulin delivery. Chem Soc Rev 43(10): 3595-3629;2014

178.   Sachin Chhajed, Sagar Sangale and S.D. Barhate; Advantageous nasal drug delivery system-international journal of pharmaceutical sciences and research 28 May, 2011

179.   Sayani AP¹, Chien YW. Systemic delivery of peptides and proteins across absorptive mucosae.1996

180.   Yıldız Ozsoy, Sevgi Gungor and Erdal Cevher, Nasal Delivery of High Molecular Weight Drugs, 21 July 2009; in revised form: 14 September 2009 / Accepted: 17 September 2009 / Published: 23 September 2009

181.   Vamshi Krishna T., Ganesh M., Madhusudan Rao Y; Formulation and Evaluation of Extended Release Non-Invasive Drug Delivery System for Insulin;Research Journal of Pharmacy and Technology ;  0974-360X;Volume No:4, Pages :  1418-1421, 2011

182.   Virginia Brown, Fang Liu. Intranasal Delivery of a Peptide with Antidepressant-like Effect. Neuropsychopharmacology,; DOI: 10.1038/npp.2014.61; 2014.

183.   Spiegelmen AR. Treatment of diabetes with synthetic vasopressin. J Am Med Assoc.  184:657–8;1983.

184.    Morimoto K, Morisaka K, Kamada A. Enhancement of nasal adsorption of insulin and calcitonin using polyacrylic acid gel. J Pharm Pharmacol; 37:134–6; 1985.

185.   Wuthrich P, Martenet M, Buri P. Effect of formulation additives upon the intranasal bioavailability of a peptide drug: Tetracosactide (ACTH1-24) Pharm Res.; 11:278–82;1994.

186.    Dyer AM, Hinchcliffe M, Watts P, Castile J, Jabbal-Gill I, Nankervis R, et al. Nasal delivery of insulin using novel chitosan based formulations: A comparative study in two animal models between simple chitosan formulations and chitosan nanoparticles. Pharm Res.; 19:998–1008;2002.

187.   Illum L, Fisher AN, Jabbal-Gill I, Davis SS. Bioadhesive starch microspheres and absorption enhancing agents act synergistically to enhance the nasal absorption of polypeptides. Int J Pharm.; 222:109–19;2001.

188.    Szewczuk MR, Campbell RJ, Jung LK. Lack of age-associated immune dysfunction in mucosal-associated lymph nodes. J Immunol.; 126:2200–4;1981.

189.   Bernkop-Schnurch, Scholler S, Biebel RG. Development of controlled drug release systems based on thiolated polymers. J Control Release.; 66:39–48;2000.

190.   Ugwoke MI. Leuven, Belgium: Leuven University Press;. Development and Evaluation of Controlled Release Nasal Drug Delivery Systems of Apomorphine;1999.

191.    Malcolmson RJ, Embleton JK. Dry powder formulations for pulmonary delivery. Pharm Sci Technol Today. ; 1:394–8;1998.

 

 

 

 

 

Received on 04.10.2018           Modified on 19.11.2018

Accepted on 16.12.2018         © RJPT All right reserved