Original Research: Surface-engineered nanoparticles for targeted delivery of therapeutic peptides to brain tissue

2.  The global market for drugs for the central nervous system
(CNS) is greatly under-penetrated, and would have to grow
by >500% just to equal the cardiovascular drug market.
 More than 98% of the newly developed agents for CNS do not
cross BBB.
 Molecules should possess low molecular weight (<500 Da) and
high lipophilicity in order to cross blood-brain barrier (BBB).
 There are only a few diseases of the brain that are currently
treated by CNS drugs.
› Only affective disorders, insomnia, pain, and epilepsy respond to
small molecules
› Most other brain diseases such as Alzheimer’s,Parkinson’s,Brain
Cancer, Stroke, Neuro-AIDS etc do not respond to small
molecules.
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3.  Recently developed large molecules such as therapeutic
proteins, peptides, genes, monoclonal antibodies,
cannot cross BBB.
 Development of drugs for brain is incomplete without a
parallel approach in brain drug delivery.
Ref: Pardridge, W.M. Brain Drug Targeting: The Future of Brain
Drug Development, Cambridge University Press, 1, (2001), pp 3-
9.
3

4.  Oral bioavailability of proteins and peptides is severely
limited due to the epithelial barriers of the GIT and
degradation by digestive enzymes.
 GI passage of particles can be achieved by formulation
of fine size ranges of approximately below 200 nm.
 A Nano-size range favors uptake through
› Absorptive Enterocytes
› Intestinal M cells
 Also, a size range below 200 nm favors escape from
spleenic filtration effects thereby enhancing circulation
half-life of nanoparticles.
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5.  Polymeric nanoparticles can allow loading of such molecules
within a polymeric matrix, protecting from enzymatic
degradation and hydrolysis, as well as targeting to brain
tissue.
 However, application of nanoparticles as oral drug delivery
systems are restricted due to their
› Limited absorption across GIT.
› Short circulation half-life.
(The peptide drug chosen for this study is dalargin, a
hexapeptide with amino acid sequence Tyr-D-Ala-Gly-Phe-
Leu-Arg which does not cross the BBB. It is an Leu-Enkephalin
analog which binds with opioid receptors in brain and causes
central analgesia)
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6.  Nanoparticles can be “double-coated” with a
combination of Tween 80 and PEG 20,000 successively to
achieve “stealth” targeting properties.
Role of Tween 80
 A Tween 80 coating over a polymeric nanoparticle leads
to the adsorption of Apo lipoprotein E (Apo E) from
plasma upon the nanoparticle surface.
 Such nanoparticles interacts with Low Density
Lipoproteins (LDL) receptors in BBB and reaches the brain
interior by endocytic uptake mechanism.
 Nanoparticles degrade in brain interior and peptide is
released.
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7. Role of PEG 20,000
 Poly (ethylene) glycol (PEG) is known to protect labile
drugs against enzymatic degradation in GIT.
 Higher molecular weight PEGs such as PEG 20,000, forms
a protective “brush” against the digestive enzymes.
 High molecular weight PEGs also provides “dysopsonic”
effect against macrophageal clearance in the blood
circulation.
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8. 8

9. 9

10.  Emulsion (Anionic) polymerization
 Medium (pH 2.5, 0.01N HCl) containing Dextran 70 (1.5%) as
an emulsifier.
 Polymerization for 4 hours with constant magnetic stirring at
8,000 rpm.
 Medium was neutralized using 0.1 N NaOH until the final pH
reached 7.0
 Size excluded by multi-filtration steps using successive filters of
5 µ, 1.2 µ and 0.7 µ pore size diameters.
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11.  Unreacted monomers and agglomerations removed by 3 cycles
of washing and ultracentrifugation at 76,500 g for 1 hour.
 Nanoparticles collected as wet pellets.
 Immediately kept in lyophilizer at -40ºC and 130 × 10-4
mbar for 12
hours for freeze drying.
 Finally, nanoparticles were obtained as free flowing, white
powder and stored at 4ºC for further use.
 The nanoparticle yield was 23% w/w.
 Entrapment efficiency (EE %) was found to be 39.84 ± 4 % w/w
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12. 12
Formulation
Codes:
Coatings with:
Tween 80 (%)
Coatings with:
PEG 20,000 (%)
T0P0 0 0
T2P0 2 0
T1.5P0.5 1.5 0.5
T1P1 1 1
T0.5P1.5 0.5 1.5
T0P2 0 2
T2P2 2 2

13. 13
 An average diameter of 100 nm with a polydispersity index of 0.018
obtained for the optimum formulation.

14. -25
-20
-15
-10
-5
0
T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2
PBCA-NDS Formulations
ZetaPotentials(mV)
14

15. 0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
Time (hours)
%DrugRelease
T0P0
T2P0
T1.5P0.5
T1P1
T0.5P1.5
T0P2
T2P2
15

16. 16
0
10
20
30
40
50
60
70
80
90
100
T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2
PBCA-NDSFormulations
%DrugRemaining
40
50
60
70
80
90
100
0 0.5 1 1.5 2 2.5 3
Time (hours)
%DrugRemaing
T2P2
T0P0

17. 0
10
20
30
40
50
60
70
80
90
100
T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2
PBCA-NDSFormulations
%DrugRemaining
17
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12
Time (hours)
%DrugRemaining
T2P2
T0P0

18. 18
Formulation Code Summary
C1 PBS solution {Control 1}
C2 PBS + Tween (2%) {Control 2}
C3 PBS + PEG (2%) {Control 3}
C4 PBS + Tween (2%) + PEG (2%) {Control 4}
C5 PBS + Drug {Control 5}
C6 PBS + Drug + Tween (2%) {Control 6}
C7 PBS + Drug + PEG (2%) {Control 7}
T2P2-N PBS + Drug + Tween (2%) + PEG (2%) + No nanoparticles present
T0P0 PBS + Drug + Nanoparticles + Tween (0%) + PEG (0%)
T2P0 PBS + Drug + Nanoparticles + Tween (2%) + PEG (0%)
T1.5P.5 PBS + Drug + Nanoparticles + Tween (1.5%) + PEG (0.5%)
T1P1 PBS + Drug + Nanoparticles + Tween (1%) + PEG (1%)
T.5P1.5 PBS + Drug + Nanoparticles + Tween (0.5%) + PEG (1.5%)
T0P2 PBS + Drug + Nanoparticles + Tween (0%) + PEG (2%)
T2P2 PBS + Drug + Nanoparticles + Tween (2%) + PEG (2%)
T2P2 + A PBS + Drug + Nanoparticles + Tween (2%) + PEG (2%) + Naltrexone HCl

19. 19
0
20
40
60
80
100
0 15 30 45 60 75 90 105 120
Time Points (mins)
%MPE
T2P2
T2P2+Anta
T0P2
T.5P1.5
T1P1
T1.5P.5
T2P0
T0P0
T2P2-Nano
(*) = p < 0.05, compared with T0P0
*
*
*

20. 0
20
40
60
80
100
7.5 15 22.5 30 37.5 45 52.5
Dose (mg/kg)
%MPE
20

21.  Double-coated PBCA-NDSs with overcoats of Tween 80
and PEG 20,000 represent a feasible method to deliver
and target peptides to brain via the oral route.
 Coating of nanoparticles with 2% Tween and 2% PEG
represents the optimal formulation for PBCA
nanoparticulate system.
 PBCA-NDSs with average diameter of 100 nm represents a
satisfactory size for oral and targeted peptide delivery to
the brain.
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