s targeted!nanopar · i e s for p, c es d b s peo oh + ch 3 o ε-cl peo noh + n 3 ε-cl 3 synthesis...

20
INSTITUTE FOR POLYMERS, COMPOSITES AND BIOMATERIALS Synthesis of func.onalized amphiphilic copolymers for the realiza.on of mul. targeted nanopar.cles by M. Malinconico 1 , G. Dal Pogge1o 1 , A. Romanelli 2 , F. Quaglia 2 , P. Laurienzo 1 1 Ins&tute for Polymers, Composites and Biomaterials, IPCBCNR, Pozzuoli 2 Department of Pharmacy, University of Naples Federico II, Naples, Italy email: [email protected]

Upload: others

Post on 16-Mar-2020

6 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

 Synthesis  of  func.onalized  amphiphilic  copolymers  for  the  realiza.on  of  mul.-­‐

targeted  nanopar.cles    by  

 M.  Malinconico1,  G.  Dal  Pogge1o1,  A.  Romanelli2,  F.  Quaglia2,  P.  Laurienzo1  

 1  Ins&tute  for  Polymers,  Composites  and  Biomaterials,  IPCB-­‐CNR,  Pozzuoli    2  Department  of  Pharmacy,  University  of  Naples  Federico  II,  Naples,  Italy    

e-­‐mail:  [email protected]    

Page 2: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

                                                   coordinated  by:  

Drug  Delivery  Laboratory,  Department  of  Pharmacy,  University  of  Napoli  Federico  II,  Naples,  Italy  

 in  collabora4on  with:  

Department  of  Biology,  University  of  Padova,  Padua,  Italy  InsItute  of  Biostructure  and  Bioimaging,  CNR,  Naples,  Italy  

IG  2014  15764  

The activities are performed in the framework of the project “Strategies to drive chemotherapeutics to solid tumors: multifunctional nanoparticles from tailor-made block copolymers” granted by AIRC-2014.

Page 3: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

+PEO OH

ε-CL CH3O

+PEO OH

ε-CL N3 N3

Synthesis of functionalized self-assembling amphiphilic copolymers

“Click” Chemistry

PEO-PCL

NPs decorated with peptides containing ‘’CendR’’ motif are able to accumulate at tumor level as they undergo transport pathway in vascular endothelial cells of tumor vessels

Page 4: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐ -­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

+++++++

-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐-­‐-­‐-­‐-­‐-­‐-­‐

+++++++-­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐

Active targeting

Vascular targeting

Passive targeting

Our   general   strategy   is   to   design   double-­‐targeted   NPs  exposing  on  the  surface  both  Tumor  Penetra&ng  Pep&des  and   folate   groups   binding   folate   receptor,  which   is   over-­‐expressed  on   the  vast  majority  of  cancer  &ssues  while   its  expression   is   limited   in  healthy  &ssues.  Furthermore,  NPs  slowly  release  their  drug  cargo  in  the  body  once  homing  at  tumor  level  is  achieved.  

• Biomime&c  • Enhanced  transport  through  tumor  vasculature  and  improved    distribu&on  inside  tumor  inters&&um  through  decora&on  with  RGD  and  CendR  mo&fs    

• Enhanced  accumula&on  in  cancer  cells  through  folate  decora&on  

• Sustained  docetaxel  release  in  tumor  inters&&um  

AIM  

Page 5: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry
Page 6: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry
Page 7: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

Amphiphilic  diblock  copolymers  

Page 8: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

Strategy  

o  PCL-­‐b-­‐PEG  with  op&mal  block  length  and  hydrophilic/lipophilic  balance  providing  NP  stability  in  the  blood  and  long  circula&on;  

o  PCL-­‐b-­‐PEG-­‐TPP  modified  at  PEG  site  through  covalent  linkage  of  already  known  or  novel  pepIde  sequences  that  promote  specific  transport  of  NPs  through  tumor  vasculature  and  accumula&on  in  the  tumor;    

o  PCL-­‐b-­‐PEG-­‐Fol  modified  at  PEG  site  with  folate  that  promote  NP  uptake  in  cancer  cells  via  receptor-­‐mediated  endocytosis.  

PCL  

PEO  

PepIde  (NGR,  CendR)  

Folate  

PEG2000-­‐b-­‐PCL4000    

Fol  –PEG2000-­‐b-­‐PCL4000  

TPP–PEG2000-­‐b-­‐PCL4000  Docetaxel  

Page 9: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

DBLS/DBL-Fol

DBLL/DBL-Fol PCL-PEGL PCL-PEGS

DBLS DBLL

PCL-PEGS +PCL-PEG-Fol*

PCL-PEGS +PCL-PEG-Fol*

* 4:1 wt ratio

PCL4.7-PEG1.5-Fol

PCL4.7-PEG1.0

PCL4.7-PEG2.0

PCL-PEGS

PCL-PEGL

PCL-PEG-Fol

Om

OO

O

OHn

CH3

OHO

NH

O

NH

N

ONH

NH2N

NO

HNN

NN

m

OO

O

OHn

Page 10: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

PCL-PEG1000

PCL-PEG1500-Fol

PCL-PEG2000

PCL-PEG1500-Fol

DBL1000 DBL2000

Folate  exposi&on  

Page 11: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS Main «blocks» synthesis  

mPEG1000-OHO

O

mPEG1000-PCL4000

N3-PEG1500-OHO

O

N3-PEG1500-PCL4000

Page 12: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

Step ISO

OCl S

O

O

NaN3

Step II

O

O

Step III

OH-PEG1,5k-OH O-PEG1,5k-OH

SO

OO-PEG1,5k-OH N3-PEG1,5k-OH

N3-PEG1,5k-OH N3-PEG1,5k-PCL4k-OH

HETERO BUILDING BLOCK

Ag2O, KI

Page 13: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

TAGS

FOLIC  ACID  

PEPTIDE  

Page 14: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

FOLATE HPLC MASS

O OH

NH

O

NH

NO

HN

H2N N N

O

OH

NH2

O OH

NH

O

NH

NO

HN

H2N N N

O

HN

O

NH

O

NH

NO

HN

H2N N N

O

OH

NH

γ

α

propargylamine

X

Page 15: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

PCL-PEG-FOL SYNTHESIS O OH

NH

O

NH

NO

HN

H2N N N

O

NH

O OH

NH

O

NH

NO

HN

H2N N N

O

HN

N NN PEG1,5k-PCL4k-OH

α

γ

N3-PEG1,5k-PCL4k-OHγ

α

N3-­‐PEG1.5k-­‐PCL4k  FOL-­‐PEG1.5k-­‐PCL4k  

_

Page 16: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

TEM image of a single DBLS/DBL-Fol SEM images of DBLS/DBL-

Fol freeze-dried

NPs were prepared by solvent diffusion of an organic phase added dropwise in water under magnetic stirring. After solvent evaporation, NPs were filtered, freeze-dried and stored at 4 °C.

Page 17: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

CONFOCAL MICROSCOPY KB cells, 1 h pre-incubation 1 mM free fol 1 h NP incubation (20 µg/ml NPs) in medium + 10% serum

PEG1000 PCL4000

Free fol + PEG1000 PCL4000 Free fol + 20% fol PEG1000 PCL4000

20% fol PEG1000 PCL4000

Effective signal reduction

Page 18: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

 Future  developments:  

POLYROTAXANE      

polymer+cd+endcapping  

Page 19: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS Why  polyrotaxane&NPs?  

Page 20: S targeted!nanopar · I E S FOR P, C ES D B S PEO OH + CH 3 O ε-CL PEO NOH + N 3 ε-CL 3 Synthesis of functionalized self-assembling amphiphilic copolymers “Click” Chemistry

INS

TITU

TE F

OR

P

OLY

ME

RS, C

OM

PO

SIT

ES

AN

D B

IOM

ATE

RIA

LS

 Synthesis  of  funcIonalized  amphiphilic  copolymers  for  the  realizaIon  of  mulI-­‐targeted  nanoparIcles    

 thanks  for  the  a:en.on!