The document presents a research proposal for developing folic acid-chitosan conjugate nanoparticles containing azithromycin for treating colorectal diseases. The objectives are to prepare drug-loaded conjugates with desired release characteristics and assess their targeting efficacy and bio-distribution. The plan involves preformulation studies, preparing the folic acid-chitosan conjugate, loading the conjugate with azithromycin, and characterizing the nanoparticles. In vitro and in vivo studies will evaluate particle properties, drug release, cytotoxicity, and the ability to treat colorectal inflammation in a rat model.

1. Department of Pharmaceutical Sciences
School of Bioscience & Biotechnology
Baba Saheb Bhimrao Ambedkar University
(A Central University)
Vidhya vihar, Raebareily Road, Lucknow-226025
Folic acid chitosan conjugate nanoparticles
containing azithromycin for the treatment of
colorectal diseases
Supervised by
Prof. (Dr.) Shubhini A. Saraf
Head Dept. of Pharmaceutical sciences
BBAU, Lucknow
Presented by
Shivam Kumar Gupta
M.Pharm
Enrollment no :566/17

2.  Introduction
 Drug profile
 Component used
 Literature review
 Research envisaged
 Plan of work
 Methodology
 Result and Discussion
 Conclusion
2

3. Introduction
Colorectal diseases result from dysregulated immune
response to intestinal microbial flora in persons with
genetic predisposition. It comprises a group of
conditions and diseases mainly colorectal cancer,
ulcerative colitis, Crohn's disease, inflammatory bowel
syndrome, diverticular disease, hemorrhoids etc.
3

4. • Colorectal cancer
• Ulcerative colitis
• Inflammatory bowel syndrome
• Crohn´s disease
• Hemorrhoids
• Bowel incontinence
• Diverticular Disease
4

5. (1) COLORECTAL CANCER
“Abnormal growth of cells continued in the same manner
after cessation of their stimuli which have initiated it with in
colon or rectum is know as colorectal cancer”.
(2) IRRITABLE BOWEL SYNDROME ( IBD)
Ulcerative colitis is an inflammatory disease of the large
intestine (the colon).
(3) CROHNS DISEASE
Also known as chronic IBD characterized by inflammation of
digestive or any part of GIT.
(4) DIVERTICULITIS DISEASE
Diverticulitis is inflammation or infection of these pockets in
the colon wall.
5

6.  Derived from Greek word Nanos which means “dwarf
or extremely small”
 Nanoparticle are solid colloidal particle size in nano
range
 Technologies that measure material or feature with at
least one critical dimension between 1nm to 100 nm
 Nanoparticle consist of micromolecular material in
which active ingredient (drug or biological active
material ) dissolved, entrapped, encapsulated ,absorbed
or attached
6

7. NANOSPHERE
(matrix type structure in
which drug is dispersed )
NANOCAPSULE (membrane
wall structure with an oil
core containing drug )
MAJOR FORM OF
NANOPARTICLE
7

8. NAME OF
COMPONE
NT
MOLECULAR
WEIGHT
MELTING
POINT
SOLUBILITY APPLICATION
FOLIC ACID 441.4 g/mol 250⁰C ETHANOL,
METHANOL
WORK AS
LIGAND
(CARRIER )
CHITOSAN 50,000 –
190,000 KDa
230-234⁰C SOLUBLE IN
DILUTE
AQUEOUS
ACIDIC
SOUTION PH ( <
6.5 )
USED AS A
POLYMER
AZITHROM
YCIN
748.996g/mol 114⁰C SLIGHT LY
SOLUBLE IN
WATER
API (MAIN
INGREDIENT )
8

9. Literature Review
Bose et al (2018) summarized in vitro and in vivo experimental
outcomes of chitosan and chitosan derivatives in anticancer therapeutic
and oral colon-specific nanocomposite carrier for colon cancer
therapeutics.
Natfji et al (2017) analyzed the polymer-drug conjugates for
inflammation to cardiovascular diseases and the rationales and
highlighted the design features to be considered when applying
polymer-drug conjugates to these new therapeutic areas.
Gumustas et al (2017) studied the effect of polymer-based
nanoparticles on the assay of antimicrobial drug delivery systems and
there effectiveness and in vitro/in vivo performance of various
antibiotics encapsulated polymer-based nanoparticles in the treatment
of infectious diseases.
Wang et al (2015) prepared and characterized tumor-targeted folic
acid–chitosan conjugate nanoparticles loaded with mitoxantrone.
9

10. Research Envisaged
• Colorectal diseases comprise a group of conditions and diseases
mainly colorectal cancer, ulcerative colitis, Crohn disease,
inflammatory bowel syndrome, diverticular disease, haemorrhoids
etc.
• As the symptoms closely resemble each other, there is potential for
misdiagnosis and mistreatment.
• Studies have shown that patients treated for colon and rectal
diseases survive colorectal cancer.
• Folic acid receptors are widely expressed in cancer as well as other
inflammatory conditions, therefore the conjugate aims to target
these receptors.
• Azithromycin has been selected as it can be used to target the
various microbes which increase inflammation during colorectal
diseases and enhance the symptoms.
10

11. OBJECTIVES
Major
 To prepare drug loaded conjugates with desired release
characteristics.
 To assess the bio distribution of drug loaded
conjugates.
Minor
 To assess the targeting efficacy of drug loaded
conjugates
11

12. PLAN OF WORK
1. Preformulation studies
•Identification of drug
•Physical appearance
•Melting Point
•Solubility analysis
•UV spectroscopy
•FTIR
2. Preparation of folic-chitosan conjugate
3. Formulation of drug loaded nanoparticle
conjugates by DOE
12

13. 4.Characterization of nanoparticle conjugate
•Drug content
•Drug entrapment
•Particle size and zeta potential
•In-vitro studies
•In-vivo studies
5. Stability studies
6. Computation and compilation
13

14. Physical parameters Azithromycin
Colour White
Odour odorless
Physical form amorphous
14

15.  Capillary Tube Method: The drug was filled from one
end of the sealed capillary and inserted into the cavity
of melting point apparatus. Temperature was observed
from the point when the drug started to melt till it got
completely melted.
 Azithromycin –Melting range is 111-1140C
15

16. Solvent Solubility as per IP
Water Slightly soluble
0.1 N HCl Slightly soluble
Ethanol Soluble
Methanol Soluble
Phosphate buffer pH 6.8 Slightly Soluble
16

17. 17

18. 18

19. y = -0.0198x + 0.239
R² = 0.9916
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8 10 12
Absorbance
Concentration (ug/ml)
Standard curve of Azithromycin in Potassium
permanganate
19

20.  Folic acid : folic acid receptor is highly expressed receptor
mainly in cancer as well as other inflammatory conditions
associated with colon disease because being a water
soluble vitamin it is also plays important role in cell repair
 Chitosan :Because of its cationic nature it is an attractive
candidate for therapeutic applications in colorectal disease .
Other reasons include it being:
 nontoxic
 biodegradable
 obtained from renewable source
 biocompatible
20

21. Role of chitosan as anticancer agent
 It also has an antitumor role through improving the body’s immune
function
 Inhibit cell metabolism which in turn inhibit cell growth finally induce cell
apoptosis
 Diet containing chitosan could reduce the generation of precancerous
lesions in colon cancer induced by azomethane compounds
21
Chitosan nanoparticles indicated that inhibition rate of 500 mg/L
chitosan nanoparticles was
 27% on Hela cells of cervical cancer,
 23% on liver SMMC-7721 cells,
 29% on gastric cancer BGC-823 cells,
 and as high as 55% on breast cancer MCF-7 cell

22. 22
FIG4: ANTI TUMOR ACTIVITY OF CHITOSAN NANOPARTICLE

23. Preparation of folic acid-chitosan conjugate:
Step1: Folic acid was dissolved in aqueous solution containing 10
mm tris HCl buffer
Step2: Chitosan was dissolved in acidic solution (0.1M HCl) and
diluted to various concentration using 10mm tris HCl
Step3: Folic acid chitosan conjugates were prepared by the addition
of different chitosan concentrations ( 1 to 60 micro molar solution ) to
acid solution
23

24. Formulation of drug loaded nanoparticle conjugate
Step1: Accurately weighed amount of drug (50-150mg)was dissolved in 2ml of
dilute hydrochloric acid solution.
Step2: Drug solution was added to conjugate and kept aside for one hour for
swelling and loading.
Step3: Glutaraldehyde was added to the above solution with sonication for 5
mins at 0.6 cycle and 60% amplitude.
Step4:Prepared formulation was centrifuged at 3000 rpm for 1 hr and NPs were
collected.
Step4: The prepared nanoparticle formulation was analysed for particle size,
PDI, zeta potential and drug entrapment.
24

25. EVALUTION PARAMETER
25

26.  Triturating 1% w/w of azithromycin in potassium
Bromide (KBr) so as to prepare pellet using hydraulic
press
finally analyzed for FTIR spectroscopy at USIC
facility, BBAU, Lucknow
26

27. Spectra obtained by FTIR Spectroscopy
Fig1: IR SPECTRA OF FOLIC ACID SAMPLE
Fig2: IR SPECTRA OF CHITOSAN
27

28. Fig3: IR SPECTRA OFAZITHROMYCIN
28

29. Fig 4: IR SPECTRA OF FOLIC ACID CHITOSAN CONJUGATE
Fig5: IR SPECYRA OF DRUG LOADED FOLIC ACID
CHITOSAN CONJUGATE
29

30. Formulation of drug loaded nanoparticle conjugates by
DOE
 On the basis of preliminary studies, the following
parameters were selected
Independent factors(high and low values)
 Folic acid conc. 40µM(-1), 80 µM(1)
 Chitosan conc. 50µM(-1), 100 µM(1)
 Amount of drug 50mg(-1), 150mg(1)
30

31. S. No. Chitosan Folic acid Drug Size (nm) PDI
Drug
entrapment
1. 1 (100µM) 1(80µM 1(150µM) 876.7 0.288 28.8
2. 1(100µM) -1(40µM) -1(50µM) 698.4 0.265 82.5
3. -1(50µM) 1(80µM) 1(150µM) 481.8 0.259 25.9
4. 1(100µM) 1(80µM) -1(50µM) 765.6 0.325 32.5
5. -1(50µM) 1(80µM) -1(50µM) 491.9 0.262 26.2
6. -1(50µM) -1(40µM) 1(150µM) 475.1 0.25 25
7. 1(100µM) -1(40µM 1(150µM) 733.2 0.373 37.3
8. -1(50µM) -1(40µM) -1(50µM) 479.5 0.206 20.6
9. 0(75µM) 0(60µM) 0(100µM) 430 0.245 15
31

32. Cumulants Results
Diameter(d):698.4 nm
Polydispersity Index(P.I.):0.265
Particle size and zeta potential
32

33. Scanning Electron Microscopy
33

34. INVITRO STUDY
34

35.  In vitro release study was carried out by using dialysis BAG
MOLECULAR WEGHT CUT OFF12-14 kDa) diffusion TECHNIQUE
PROCEDURE:
2 ml of formulation
poured into dialysis bag then placed into a beaker
containing 50ml of PBS AT 37+2C with magnetically stirred at
50 rpm at predetermined time interval for 24 hr
1ml of sample was withdrawn and sink condition was
maintained by replacing with fresh PBS of same temperature
Finally sample was suitably diluted and analyzed by UV
Visible spectrophotometer
35

36. 0
20
40
60
80
100
120
0 5 10 15 20 25 30
%
cumulative
drug
release
Time in hours
percent release in 0.1 N HCl, pH 7.4 and pH 9.2
Drug release in 0.1 N HCl 2hr, pH 7.4 upto 5th hr, pH 9.2 upto 24th
hr
36

37. Cell line
IC 50 studies on A549 cells
37

38. IN VIVO STUDY
38

39. Rat are sedated with I.P (Intraperitoneal ) injection of
phenobarbitone (35mg/kg)
+
3ml acetic acid induced with polyethylene tube were
placed in rectum for 30 sec and then fluid was
withdrawn
finally animal was weight and check for diarrhea
39

40. Add 3 ml of acetic acid in 97ml of distilled water
Inserted into rat through Intraperitoneal injection in rat
finally cause ulcerative colitis in rats
40

41. (A) MACROSCOPIC SCREENING:
Colon are excised and opened longitudinally
Rinsed with cold saline and colonic damage was evaluated by using miller scale
MILLAR SCALE
0-NORMAL
1-MUCOSAL ERYTHEMA
2-MILD BLEEDING
3-MODERATE BLEEDING
4-SEVEREULCERATION
41

42. All animal are sacrifice after 5 day of inducing colitis
Colon removed and lumen washed with 3ml prereduced sterilized
scheduler broth Prepare slide in which specimen is stained with
Haematooxylin and Eosin and Alcan blue
Different slide are coded with different number so as to prevent the
observer bias
All slide are observed under Olympus BH-2 microscope
42

43. Tissue specimens for histopathology
Control Colitis specimen Treatment
43

44. 44
FIG1(A )H&E CROP 100 REPRESENT Section of colon showing
marked infiltration of mononuclear cells i.e. lymphocyte in
mucosa,submucosa & the deeper layer of the intestine
degeneration & deformation of epithelial cell along with goblet cell
hyperplasia
FIG1(B) H&E CROP 400REPRESENT Higher magnification of fig1(a)

45. 45
FIG 2(A) H&E CROP 100 REPRESENT Degeneration section of
colon showing necrosis tissue in the lumen indicating
degeneration of epithelial cell as well as of mononuclear cell as
well as goblet cell hyperplasia
FIG 2(B) H&E CROP 400 REPRESENT higher magnification of fig
2(b)

46.  J
46
FIG 2(A) H&E CROP 100 REPRESENT Mild degeneration as well as
desquamation of epithelial cell as well as mild infiltration of
mononuclear cell in mucosa whereas partial recovery of lamia
muscularis of mucosa
FIG 2(B) H&E CROP 400 REPRESENT Higher magnification of figure
2(A)

47. G
47
FIG4.: H&E CROP 100 REPRESENT Majority of enteric glands are
normal whereas there is normal infiltration of mono nuclear in
mucosa Layer of colon

48. 48
Conclusion
 By incerasing the concentration of chitosan loading capacity of
drug increases
 Being water soluble vitamin B9 along with chitosan play
important role as an anticancer agent in colon targeting
diseases by improving immune system of the body therefore
provide synergistic effect along with azithromycin
 Optimized formulation were characterized for particle size,
zeta potential, poly dispersibility index, drug entrapment ,drug
release, FTIR for functional group detection as well as shape and
morphology was evaluated by using scanning electron
microscopy (SEM) study reveals that most of the particle present
in a formulation were spherical in shape.

49. 49
 In conclusion it is assumed that f2 showed better release
pattern, greater entrapment efficiency, which is beneficial
for future drug formulation perspective.
 various parameter of optimized formulation involved
s.no Parameter Result
1. Particle size 698.4nm
2. Poly dispersibility index (PDI) 0.265
3. Zeta potential 25.96
4. Entrapment efficiency 82.5%
5. Dissolution in 0.1N HCL,
phosphate buffer(pH 7.4and 9.2)
Better sustained
effect for 24 hour
In vivo studies were also carried out to assess the targeting
ability and efficacy of the formulation. Histopathological
data shows that the formulation effectively treats acetic
acid induced colitis.

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