PHARMACOKINECTIC AND

PHARMACODYNAMIC ASPECTS OF
HUMAN SERUM ALBUMIN & BOVINE
SERUM ALBUMIN NANOPARTICLES IN
CANCER THERAPY

By
Kinshuk Bhojwani
U. Harini Goud
Contents
• Introduction
• Types of Albumin
• Human Serum Albumin
Preparation Of HSA-NP
PK-PD Of HSA-NP
• Bovine Serum Albumin
Preparation Of BSA-NP
PK-PD Of BSA-NP
• Conclusion
 INTRODUCTION
• Cancer comprises a variety of diseases that ascend as a result of the unregulated
progress of malignant cells, which have the potential to conquer or spread to other
body parts.
• Albumin based nanoparticles are intended to improve the delivery effectual to the
tumour site.
• Albumin is a versatile material to formulate nanoparticles for drug delivery, due to it
high binding capacity of drugs and decent biocompatibility.

Human serum albumin (HSA) Bovine serum albumin (BSA)

high solubility in water high solubility in water
extensive half-life in blood extensive half-life in blood
molecular weight (65-70 kDa) molecular weight (65-70 kDa)
585 amino acids 583 amino acids
• This review discusses:

the formulation and preparation of nanoparticles utilizing human serum

albumin and bovine serum albumin, and

the pharmacokinetics and pharmacodynamics of nanoparticles.

There are two types of albumin:
Human serum albumin(HSA)
Bovine serum albumin(BSA)

These serum albumin proteins are obtained from human serum and cow serum

HUMAN SERUM ALBUMIN:-

• HSA is the most abundant protein in the human body with a MW of 66.5 kDa,
produced by the liver and has a half-life of 19 days.
• By X-ray structure analysis, the structure of HSA comprises of three domains-
I, II and III.
• These domains consists of two sub domains each -Ia, Ib, IIa, IIb, IIIa and IIIb,
which are arranged together to form binding sites on the HSA molecules.
• HSA can bind to metabolic substrates, therapeutic drugs, which include
hydrophobic as well as hydrophilic drugs.
• HSA has target specific site for the glycoprotein60 receptor present on the
surface of cancer cells, which allows the delivery of various anti-cancer drugs,
such as docetaxel, paclitaxel
BOVINE SERUM ALBUMIN:-
 Bovine serum albumin could be substituted by human serum albumin ,having a molecular weight of
69,323 Da and an isoelectric point (pI) of 4.7 in water (at 25 °C)
 Widely used for drug delivery because of its medical importance, abundance, low cost, ease of
purification, and its wide acceptance in the immunologic response in vivo.

These are used in the production of nanoparticles such as HSA nanoparticles and BSA nanoparticles.
They share similar properties as;
 High solubility in water
 Long half life in blood
 Molecular weight ( 65-70k Da)
 Similar number of amino acid residues
 HSA (585)
 BSA (583)

The main difference between two proteins from the spectroscopic point of view is that BSA has two
tryptophan residues ( W 131 and W214) while HSA has only one tryptophan (W214) .
CASE STUDY OF HUMAN SERUM ALBUMIN NANOPARTICLE[3]

• PREPARATION OF F-CM-HSA-NP’s
1. Preparation of curcumin based hsa-np’s(CM-HAS-NP’s)
2. Preparation of folate-conjugated, curcumin human serum albumin nanoparticles (F-CM-HSA-NP’s)

 PREPARATION OF CURCUMIN BASED HSA-NP

(ZHIWANG et al,)

900 mg of HSA was dispersed in
25 mL water which is saturated
with chloroform (Solution A)
This results in the formation of o/w
emulsion and homogenized for ten
cycles at 20,000 psi.
Then 100 mg of
the Solution B was
Curcumin was
added drop by drop
dissolved in 1.5 mL
into the Solution A was
chloroform saturated
with water (Solution
stirred at the rate of
300 rpm
B)
The formed emulsion The nanoparticles thus formed
was rotary evaporated were filtered through a
membrane syringe filter and the
for 20 minutes to allow solvent was removed by
evaporation of lyophilization for 36 hours at
−70°C. The powder of CM-
chloroform at 25°C, HSANPs was collected under
under reduced pressure vacuum for 24 hours at 4°C
PREPARATION OF FOLATE-CONJUGATED, CURCUMIN HUMAN SERUM ALBUMIN
NANOPARTICLES

Why folate conjugation?

It selectively targets the tumor tissues (folic acid)
Small size, low immunogenicity , low cost ,solvent compatibility ,storage stability and high affinity

For the preparation of F-CM HSANP’s

5 mg of NHS-folate (N-HYDROXY SUCCINAMIDE ESTER OF FLOIC ACID) dissolved in 1 mL of

carbonate buffer solution (0.2 M, pH 10) was added dropwise to the CM-HSANPs (2 mL) with gentle
stirring. This process was allowed to proceed for 1 hr at room temperature.
Above obtained liquid was centrifuged at 12,000 rpm for 20 minutes and simultaneously washed with
buffer solution of bicarbonate. This results in the formation of NPs which were then freeze dried without
any other cryoprotectant
 PHARMACOKINECTIC PROFILE OF CURCUMIN LOADED
HUMAN SERUM ALBUMIN NANOPARTICLES

For the study of pk aspects of F-CM-HAS-NPs ,the rats of

weight ranging between 200 g and 250 g used

Earlier than the experiment ,These rats kept under fasting conditions for 1
day .
three groups of rats (6 per batch) were administered via i.v by tail vein with
either curcumin solution (10 mg/kg, as a control) or C-HSANPs and F-CM-
HSANPs formulation.

at different time intervals(0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24

hours),2 mL of serum samples were collected in pressure
equalization tubes ,before and after drug administration. The
resulted sample of serum were frozen at −20°c until the
analysis of hplc is performed.
•The pharmacokinetic studies of CM solution, CM-HSANPs,
and F-CM-HSANPs were performed in the rats (Figure 1).

• Based on the obtained results, in the group of CM solution, in

the first 5min high concentrations of free CM in the plasma
were recorded after dosing; after 6 hours its concentrations
reduced rapidly and became undetectable.

• On the contrary the concentrations of CM encapsulated in

HAS-NPs could still be determined at 24 hours following
injection.
Figure 1- Plasma concentration–time profiles
•The Pk results indicated that the clearance of CM
of CM solution, CM-HSANPs, and F-CM-
encapsulated in NPs decreased, and preparation of CM into HSANPs after IV administration to rats (n=6).
NPs could appreciably change the pharmacokinetic behavior of
CM.
•The elimination half life( t1/2) values of CM-HSANPs and F-CM-HSANPs were recorded
as 0.36 hrs and 0.43 hrs(figure 2)
• when compared to free CM ,nanoparticles provided higher AUC0–∞ (3-fold) and half-life
(5-fold).
•therefore, ultimately that nanoparticles could significantly expand the role of Cur cumin in
vivo which resulted in improved bioavailability.
This is mainly because a lot of HSA blocks were conjugated to the surface of the
Nanoparticles, which could significantly increase the circulating time of Nanoparticles in
the blood.

Figuere 2-Pharmacokinetic parameters of CM after IV administration of CM

solution, CM-HSANPs, and F-CM-HSANPs to rats (n=6)
PHARMACODYNAMIC STUDY
IN VIVO ANTITUMOR ACTIVITY

i. BALB/c male mice which were about 6–7 weeks old were used as a tumor xenograft model.
ii. Human colorectal adenocarcinoma 29 cells(5×108 cells/mice, 50 μL injection)were
subcutaneously inoculated into a dorsal flank of each mouse to establish tumors.
iii. 5 days post tumour cell inoculation, the mice were given phosphated-buffered saline, free
CM, CM-HSANPs, and F-CM-HSANPs dispersed in normal saline every alternate day for 10
days.
iv. To measure the tumor diameters, and tumor volumes (in mm3) an instrument called digital
caliper was used. the following formula to determine the tumor volume (Tumor volume
=length ×Width2×0.5)
v. In order to monitor the potential toxicities, the body weights were estimated for every 2 days.
The in vivo antitumor study was terminated 20 days after injection.
To assess the antitumor action F–CM -HSANPs in colon cancer xenograft models in-vivo,
examination of the progression of tumor and changes in the body weight are done in nude
mice which are treated with saline which is free from Curcumin , CM-HSANPs, and F-CM-
HSANPs (10 mg/kg).
Tumors cells were developed at all sites post 5 days of tumor cell injection. The highest
progression rate of tumor growth was found in the mice which is treated with saline, while the
free CM was observed to have a minor antitumor activity. (18%)
However, C-HSANPs and F-C-HSANPs extensively inhibited the growth of(Human
colorectal adenocarcinoma 29-derived tumors (45% and 64%) at the 10th (Figure 3).

Figure 3 In vivo antitumor activities of free CM, CM-HSANPs, and F-CM-

HSANPs (10 mg/kg) in human colon cancer (HT29 cells) xenograft models
after the IV administration
CASE STUDY OF BOVINE SERUM ALBUMIN NANOPARTICLE [4]

PREPARATION OF BUFALIN LOADED BSA NANOPARTICLES

Huiqing Zhang et al; Prepared BUFALIN-BSANPs by desolvation technique.

• Ethanol was dropwise added to the albumin (pH 5.5) with continuous stirring until
the solution turns turbid.
• During this process, phase separation of albumin occurs, while adding ethanol to the
solution, due to albumins diminished water solubility.
• The albumin particles that are formed, their stability is not enough, therefore they
get redissolved again with water.
• Hardening of the coacervates occurs due to the condensation reaction with the
aldehyde group of glutaraldehyde between the arginine moieties and amino moieties
in the guanidino site and lysine residues of albumin.
PHARMACOKINECTIC PROFILE OF BUFALIN LOADED BOVINE
SERUM ALBUMIN NANOPARTICLES
 Huiqing Zhang et al; used six groups of Wistar rats which were
administered with a single dose of Bufalin-BSA-NP and Bufalin
solution with concentration of 0.15, 0.3, 0.6mg/kg respectively.
Bufalin plasma concentration time thus, obtained after administration
of different formulation is shown in graphs.

 The BUFALIN-BSA-NP showed a longer plasma clearance as

compared to normal Bufalin solution. The AUC, Half-Life,
Clearance Rate and Mean Residence Time[MRT] of BSA-NP was
found to be 1.19-1.81, 2.17-3.16, 0.55-0.82 and 2.12-3.61 times to
that of Bufalin solution, respectively.
Graph :(A, B, C) Bufalin concentration of Bufalin and Bufalin-BSA-
NP at doses of 0.15 (A), 0.3 (B), respectively
0.6 (C) mg/kg
TISSUE DISTRIBUTION OF BUFALIN-BSA-NP:

The concentration of Bufalin-BSA-NP and Bufalin was investigated in Wistar Rats

having walker 256 liver cancer cells.
The BUFALIN-BSA-NP was uptaken markedly in higher concentration in liver, tumor
and spleen.
The concentration of BUFALIN-BSANP was 352.045±35.665ng/gm and bufalin
solution was 164.465±48.80ng/gm after 5 min. The uptake of BUFALIN-BSANP was
higher than bufalin solution after 5mins, 15min and 45min by the tumor cells.

The relative uptake of BUFALIN-BSANP by other organs like liver, heart, kidney was
lower than the bufalin drug solution.
Therefore, the toxicity to heart kidney and lungs was significantly reduced and the
BUFALIN-BSANP was found to be more superior than bufalin because of its ability to
deliver the drug to both liver and tumour.
Graph :(D, E, F) tissue distribution of Bufalin and Bufalin-BSA-NP in
vivo at 5, 15, and 45 min after a single dose of 0.3mg/kg. Values are
expressed as mean ± standard error of mean; n = 5.
PHARMACODYNAMIC STUDY: BUFALIN-BSANP DEMONSTRATED MORE
POTENT AND TUMOUR EFFECTS THAN BUFALIN ON HEPATOCELLULAR
CARCINOMA IN NUDE MICE.
Graph :(A) Tumor volume of different groups; (B) survival analysis of different groups. ADM:
Adriamycin; NS: normal saline. Values are expressed as mean ± standard error of mean.
 After conjugation of CM-HSA-NP with folate, sustained release was observed
compared to the unconjugated NP. Also, an increase in the retention time of CM was
observed.

• The improved anti tumour activity of the F-CM-HSA-NP attributed to the protection
of drug from enzymatic deactivation followed by the selective localization at the
desired site.

• The AUC, Half-Life, Clearance Rate and Mean Residence Time[MRT] of BSA-NP
was found to be 2-3 times to that of Bufalin solution.

• The BUFALIN-BSA-NP showed extended longevity when compared with the

BUFALIN group, Adriamycin group, with the NS group.
Thank you!