NLS presentation

1. Novel approaches for targeted delivery of cancer therapeutics to nuclei of malignant cells
Animikh Ray
Scientist-C
Father Muller Research Centre
Assistant Professor
Dept. of Biochemistry
Father Muller Medical College
1

2. Synopsis
• Introduction
• Prodrug approach
• Nuclear Localization signal(NLS) approach
-peptide synthesis and purification
-uptake of peptides and colocalization with nucleus
-nanoparticle preparation and optimization
-uptake of NLS conjugated nanoparticle
• Conclusion
2

3. Introduction
3

4. 4
Nag, O.K.; Delehanty, J.B. Active Cellular and Subcellular Targeting of Nanoparticles for Drug
Delivery. Pharmaceutics 2019, 11, 543. https://doi.org/10.3390/pharmaceutics11100543
Cellular vs intracellular targeting:

5. Understanding cancer
R. Weinberg, E. Hanahan 2000 Cell 100(1) 57-70
• Evading programmed cell death
• Limitless replicative potential
• Sustained angiogenesis
• Metastasis
5

6. Significance of nuclear drug delivery
• First-line anticancer drugs are DNA toxins
acting directly on DNA or associated enzymes
• Doxorubicin- interacts with Nuclear DNA by
intercalation
• Cisplatin- Binds DNA and causes its cross
linking
6

7. Barriers for nuclear drug delivery
• Extracellular barriers
• Bioactive molecules (e.g. enzymes)
• Immune defense
• Size of the nanocarrier (< 5 nm , >200 nm are cleared or scavenge
Plasma membrane
• Intracellular Barriers
• Phagocytosis
• Vesicles (Lysosomes, Endosomes); Nuclear envelope ( Nuclear Pore complex); Nuclear transport – Substantial barrier
7

8. Doxorubicin
• Molecular formula:
C27H29NO11
• Molecular Weight – 523.5
Mechanism of action:
It intercalates with DNA and
inhibits macromolecule
synthesis.
8

9. Adverse effects
• Cardiotoxicity
• Arrhythmias
• Hypotension
• Congestive heart failure(related to total dose administered)
• Cardiomyopathy
• Bone marrow depression
• Alopecia
• Stomatitis
• Vomiting
• Local tissue damage
9

10. 10
Use of Dox in Formulation Development:
Dox is one of the most commonly studied drug for drug delivery
studies
offers several advantages for research aside from its use as an
effective anticancer drug.
 The hydrochloride form of dox is water soluble
it is fluorescent
monitored before and after formulation development

11. Prodrug approach
11

12. Translocation of pro-drug across plasma and nuclear membrane of breast cancer cells
Hypothesis:
12

13. Expression of PEPT on
plasma membrane of T47D
Uptake of [3H] glysar in the presence of unlabeled glysar, cephalosporins in T-47D
13

14. Expression of PEPT on
nucleus of T47D
Nuclear uptake of [3H] glysar in presence of unlabeled glysar
14

15. Docetaxel-dipeptide/paclitaxel-dipeptide
OH
O
O OH
OH
OH
O
O
NH2
OH
O
OH
Doxorubicin
+
i)DIEA,HBTU
ii)20%piperidine/DMF
stirring,4.5,rt OH
O
O OH
OH
OH
O
O
NH
OH
O
OH
O
Doxorubicin-dipeptide
HOCO-dipeptide dipeptide
Synthesis scheme for valine-valine dipeptide doxorubicin prodrug.
Synthetic scheme:
15

16. Uptake of Dox and L-Val-L-Val-Dox in T47D cells
Uptake of Dox and L-val-L-val-Dox in T-47D cells
16

17. Stability of L-Val-L-Val-DOX in
T47D cell homogenates
stability data of doxorubicin prodrug in T-47 D cell homogenate
17

18. Translocation of pro-drug across plasma and nuclear membrane of breast cancer cells
Hypothesis:
18

19. 19

20. Nuclear Localization signal(NLS) approach
20

21. Objective
• The primary objective of this project is to design novel nuclear
localization signal (NLS) peptide analogues that can carry therapeutic
molecules specifically into the nucleus of cancer cells.
• This strategy can reduce toxicity to healthy cells by delivering
anticancer drugs to subcellular organelle i.e. nucleus of cancer cells
preferentially.
21

22. • Nuclear localization signal (NLS) can facilitate cargo entry across
plasma and nuclear membrane
• NLS has limited application in drug delivery due to lack of specificity
towards cancer cells, which may result in toxicity
22

23. Aim of Project
• Development of an optimal NLS which will carry cargo to nucleus
with minimal toxicity to normal cells.
• Attaching the optimized NLS to nanoparticles/drugs would allow drug
delivery across nucleus.
23

24. Clinical relevance
• These novel NLS peptide analogues when conjugated to the surface of
anticancer drug loaded NP may enhance specificity and cargo delivery
inside the cancer cell nucleus.
• Such strategy can reduce toxicity to healthy cells.
24

25. NLS of choice
• Adenovirus fiber protein is an NLS and has two important sequences.
One is NLS sequence and the other is receptor mediated endocytosis
(RME or CPP)
25

26. {CKKKKKKKSEDEYPYVP}{NFSTSLRARKA}
Cell penetrating peptide (CPP) Nuclear Localization Signal
(NLS)
26

27. 27
Proposed Modifications:

28. Sequences of NLS synthesized
NLS1- CKKKKKKKSEDEYPYVPNFSTSLRARKA
NLS2- CKKKKKKKSEDEYPYVPNVSTSLRARKA
NLS3-CKKKKKKKSEDEYPYVPNVSTSVRARKA
NLS4-CKKKKKKKSEDEYPYVPNVSTSVRVRKA
NLS5-CKKKKKKKSEDEYPYVPNVSTSVRVRKV
Modifications introduced:
Phenylalanine replaced with valine
Leucine replaced with valine
Alanine replaced with valine
Alanine replaced with valine
28

29. peptide synthesis and purification
29

30. Solid Phase peptide synthesis
F-moc deprotection
coupling
30

31. NLS1 NLS2 NLS3 NLS4 NLS5
Mw 3330.9 Mw 3282.9 Mw 3268.8 Mw 3296.9 Mw 3324.9
m/z m/z m/z m/z m/z
1 3331.9 1 3283.9 1 3269.8 1 3297.9 1 3325.9
2 1666.45 2 1642.45 2 1635.4 2 1649.45 2 1663.45
3 1111.3 3 1095.3 3 1090.6 3 1099.967 3 1109.3
4 833.725 4 821.725 4 818.2 4 825.225 4 832.225
5 667.18 5 657.58 5 654.76 5 660.38 5 665.98
6 556.15 6 548.15 6 545.8 6 550.4833 6 555.15
7 476.8429 7 469.9857 7 467.9714 7 471.9857 7 475.9857
8 417.3625 8 411.3625 8 409.6 8 413.1125 8 416.6125
9 371.1 9 365.7667 9 364.2 9 367.3222 9 370.4333
10 334.09 10 329.29 10 327.88 10 330.69 10 333.49
m/Z values of NLS peptides
m/Z values
31

32. Chromatogram showing elution of NLS 1
Purification of NLS 1
32

33. UV and MS spectra of purified fraction of NLS 1
+5
+6
Purification of NLS 1
33

34. Chromatogram showing elution of NLS 2
Purification of NLS 2
34

35. UV and MS spectra of purified fraction of NLS 2
+8
+5
Purification of NLS 2
35

36. Chromatogram showing elution of NLS 3
Purification of NLS 3
36

37. UV and MS spectra of purified fraction of NLS 3
+5
+6
Purification of NLS 3
37

38. Chromatogram showing elution of NLS 4
Purification of NLS 4
38

39. UV and MS spectra of purified fraction of NLS 4
+4
+6
Purification of NLS 4
39

40. Chromatogram showing elution of NLS 5
Purification of NLS 5
40

41. UV and MS spectra of purified fraction of NLS 5
+4
+8
Purification of NLS 5
41

42. Cytotoxicity of peptides
0
20
40
60
80
100
120
Triton X Medium NLS1 NLS2 NLS3 NLS4 NLS5
%
Cell
Viability
*
MTT Assay in D407 cell line(24 hours)
42

43. uptake of peptides and colocalization with nucleus
43

44. Quantifying peptide uptake by nuclear extraction method
1 x 106 cells plated onto 12-well plates
Washed cells with PBS, and expose to 300 µL of 10 µM FITClabelled
peptides in serum free media for 30 minutes
PBS wash and trypsinize for detachment
Isolation of nucleus- sucrose gradient centrifugation 44

45. Analysis of uptake by nuclear extraction
0
50
100
150
200
250
300
CONTROL N1 N2 N3 N4 N5
D407 Y 79
ug/mg
protein
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
CONTROL N1 N2 N3 N4 N5
Specificity ratio(y79/d407)
Specificity ratio(y79/d407)
B
A and B-Specificity ratios of FITC tagged modified NLS peptides to nuclei of retinoblastoma cells and normal RPE
cells
45

46. Protocol for uptake studies for confocal imaging:
• purified FITC conjugated NLS peptides
46
Treat cells
Plasma membrane stain
20 minutes
Fix and counterstain
with DAPI nuclei stain

47. T47D
MCF10A
Y-79
D407
Glioblastoma
Astrocytes
CRL-2066
Lung fibroblast
confocal image of free FITC uptake A-DAPI,B-cell membrane stain,C-FITC D-Merge
B
A C D
Control
47

48. confocal image of NLS 1 uptake-DAPI,B-cell membrane stain C-FITC tagged NLS 1 D-Merge
T47D
MCF10A
Y-79
D407
Glioblastoma
Astrocytes
CRL-2066
Lung fibroblast
A B C D
NLS 1 uptake
48

49. T47D
MCF10A
Y-79
D407
Glioblastom
a
Astrocyte
s
CRL-
2066
Lung
fibroblast
confocal image of NLS 2 uptake-DAPI,B-cell membrane stain C-FITC tagged NLS 1 D-Merge
A B C D
49
NLS 2 uptake

50. T47D
MCF10A
Y-79
D407
Glioblastoma
Astrocytes
CRL-2066
Lung
fibroblast
confocal image of NLS 3 uptake-DAPI,B-cell membrane stain C-FITC tagged NLS 1 D-
Merge
A B C D
50
NLS 3 uptake

51. T47D
MCF10A
Y-79
D407
Glioblastoma
Astrocytes
CRL-2066
Lung
fibroblast
confocal image of NLS 4 uptake-DAPI,B-cell membrane stain C-FITC tagged NLS 1 D-Merge
A B C D
51
NLS 4 uptake

52. T47D
MCF10A
Y-79
D407
Glioblastoma
Astrocytes
CRL-2066
Lung fibroblast
A B C D
52
NLS 5
uptake

53. Co-localization
Co-localisation = Relationship between channel intensities
The presence of signal intensity (two or more labels) in the same pixel
(physical/cellular structure)
Ultimate limit: resolution of the microscope (approx. 200x200x800 nm)
53

54. Interpretations:
The values
Rr= 0.5: cutoff for colocalization
• Rr= 1 : perfect colocalization/correlation
• Rr= 0 : random (no) colocalization
• Rr= -1 : perfect exclusion/anti correlation
The Pearson’s Coefficient (PC)
54

55. 55

56. Pearson’s coefficient and correlation coefficient of peptides for different cell lines
Colocalization table
56

57. Possible mechanisms of Nuclear uptake of NLS
Nuclear protein import pathways. A. Conventional nuclear import mechanism of
cargo containing NLS mediated by the importin (Imp) α/β heterodimer. B. Nuclear
import of cargo mediated by Imp β alone.
57

58. Importins overexpressed in tumors
58

59. Docking by Z-dock
• ZDOCK is Fast Fourier Transform based protein docking program
authored and maintained by Zhiping Weng's lab (ZLAB) at
the University of Massachusetts Medical School.
• ZDOCK searches all possible binding modes in the translational and
rotational space between the two proteins and evaluates each pose
using an energy-based scoring function.
59

60. Interaction of NLS peptides with
importin alpha 2
A
C
B
D
E
NLS importin complex, Green-NLS peptide, blue
and red - importin α 2 (A) NLS 1, (B)-NLS 2, (C)
NLS 3, (D) NLS- 4, (E) NLS 5
60

61. Docking score for peptides
Docking scores
61

62. nanoparticle preparation and optimization
62

63. synthetic scheme of PLGA-PEG-NH2
Polymer synthesis
63

64. Nanoprecipitation method used for preparation of nanoparticles
Nanoparticle preparation
64

65. 0 20 40 60 80 100 120 140 160 180 200
PLGA-PEG NP 15 mg/ml acetone
PLGA-PEG NP 15 mg/ml in DMF
plga-peg NP 15mg/ml THF
plga-peg NP 15mg/ml acetonitrile
nanometer
Chart Title
Effect of different organic solvents on size of nanoparticles
Parameter optimization
65

66. 0 20 40 60 80 100 120 140
15 mg/ml
10 mg/ml
5mg/ml
nanometer
DMF
Effects of different polymer concentrations dissolved
in DMF on NP size
66
Continued:

67. 0 20 40 60 80 100 120
DMF:water 1:1
DMF:water 1:2
DMF:water 1:5
DMF:water 1:10
nanometer
Chart Title
Effects of aqueous and organic phases (DMF) ratio on the size of NP
67
Continued:

68. Name of parameters Optimization
Organic Phase DMF
Polymer concentration 5 mg/ml
organic and aqueous
phase ratio
1:2, 1:5
Optimized Parameters
68
Continued:

69. 0 50 100 150 200 250
DMF:water 1:5 1% doxorubicin
DMF:water 1:2 1% doxorubicin
DMF:water 1:5 5% doxorubicin
DMF:water 1:2 5% doxorubicin
DMF:water 1:5 10% doxorubicin
nanometer
Chart Title
Size distribution of nanoparticles prepared with different concentrations of
doxorubicin and altered ratio of DMF and water.
69
Continued:

70. Percentage of encapsulation efficiency and drug loading for
optimized formulations
70
Continued:

71. uptake of NLS conjugated nanoparticle
71

72. preparation scheme of functionalized nanoparticles
Preparation of targeted
nanoparticle
72

73. uptake of nanoparticles in a 3D model of T47D
Uptake of targeted nanoparticle
73

74. Merged images showing co-localization of Doxorubicin and DAPI
nuclear stain in a 3D model of tumor. Doxorubicin loaded nanoparticles
surface modified with NLS 3 and 5 show much greater co-localization
compared to unmodified drug loaded nanoparticle and nanoparticle surface
modified with NLS 1(native NLS).
Merged image of uptake of
targeted nanoparticle
74

75. Conclusions
• Some of the modified NLS peptides are taken up
preferentially by cancer cells.
• This has the potential to reduce toxicity associated
with conventional chemotherapy.
• These modified peptides are probably taken up by
enhanced expression of importins in tumor cells.
75

76. Future studies
• Study the interaction of synthesized peptides with different importins and cell
membrane proteins which are overexpressed in tumors
• Understand how these peptides cross plasma membrane of cancer cells
preferentially
• Synthesize modified NLS peptides with enhanced electrostatic, H-bonding and
hydrophobic interactions
• Study preferential uptake of these peptides in animal models of tumor
76

77. 77

78. 78

79. 79