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University of Massachusetts Medical School

University of Texas M. D. Anderson Cancer Center


Rice University

LMIR Lab Meeting Journal Club

Zhongli Cai
October 6, 2015

INTRODUCTION

Surface-stabilized AuNPs for theranostic biomedical


applications
hyperthermic therapy
radiosensitizer
radionanomedicine

Challenges to clinical use of AuNPs


Stability of surface coating
Adsorption of proteins to AuNPs

High uptake in the reticuloendothelial system


Intraendosomal aggregation in the cells

Ideal compound for surface coating


Denser PEG-based polymer without terminal OH
methoxy(polyethylene glycol)-polylysine conjugates
(MPEG-gPLL)

METHOXY(POLYETHYLENE GLYCOL)POLYLYSINE CONJUGATES (MPEG-gPLL)


Also called protected graft copolymers (PGC)

long
circulation
time

METHOXY(POLYETHYLENE GLYCOL)POLYLYSINE CONJUGATES (MPEG-GPLL)

Applications
a carrier system
blood pool imaging
agent

detection of local
inflammation
blood supply of
tumors

passed the initial


safety phase of
clinical trials
PGC-[99mTc]DTPA
Theranostics, 2012; 2(6):553-576

PROPERTIES OF PGC-DTPA(Gd) SYNTHESIZED USING POLY-LLYSINE HYDROBROMIDE WITH A DEGREE OF POLYMERIZATION OF

252 AND A MW(LALLS) OF 41.8 KD

Theranostics, 2012; 2(6):553-576

HYPOTHESIS

methoxy(polyethylene glycol)-polylysine
conjugates, i.e., MPEG-gPLL, have a potential
use in AuNP stabilization a plurality of amino groups

MPEG5000
Schema of water phase synthesis of AuNPs using capping/stabilizing MPEGgPLL graft copolymer and optional trisodium citrate, n = 110, m=164

AIMS

1. investigate the ability of MPEG-gPLL


to stabilize and simultaneously to

functionalize AuNPs
2. test the heating and electrical properties

of the obtained AuNP3

RESULTS
Synthesis

and

Characterization

of

MPEG-gPLL and AuNPs


Stability

and Behavior of MPEG-gPLL-

Stabilized AuNPs in Biological Systems


Evaluation

of AuNP3s for RF

Applications

Ablation

SYNTHESIS AND CHARACTERIZATION OF


MPEG-gPLL AND AuNPS

MPEG-gPLL
a PEGylation degree of
30%
~ 25% MPEGylated N-amino groups
6-7 nm
Linkage

MPEG-gPLL1: very stable


urethane bonds
MPEG-gPLL2: less stable
amide bonds with an
additional labile ester bond

MPEG-gPLL1

SYNTHESIS OF AUNPS

Image of the PCR well-plate


acquired after the synthesis of
AuNPs in the presence of
various concentrations
of
HAuCl4 and MPEG-gPLL
Fig. 1

Acceptable concentration range:


HAuCl4 : 140160 M,
the MPEG-gPLL1 >0.3 mg/mL
4

Comparative absorbance spectra showing the


positions and intensities of the GNP plasmon
peaks measured in the wells of the plate
shown in (B) with the corresponding well
series indicated on the right.

HAuCl44 : 140 M,
MPEG-gPLL : 0.9 mg/mL

TABLE 1 PROPERTIES OF AuNPS

This allowed the use of size-exclusion chromatography for purity analysis


and for purication of AuNP3s from low molecular weight impurities.

by weight : 34% gold + 66%


hydrated MPEG-gPLL1

Properties of AuNP3

MPEG- gPLL1: 67 nm,


strongly positively charged
Before
purification

After
purification

number size
distribution
Z-average hydrodynamic

9-nm-thick
polymer
layer

Fig. 2

TEM

Uranyl acetate-enhanced
contrast staining

Backscatter
(compositional) SEM

STABILITY AND BEHAVIOR OF


MPEG-gPLL-STABILIZED
AuNPS IN BIOLOGICAL SYSTEMS

STABILITY OF MPEG-gPLLS: SIZE-EXCLUSION HPLC


PROFILES OF 99mTc-MPEG-gPLL1 AND 99mTc-MPEG-gPLL2
80% mouse

99mTc-MPEG-gPLL

MPEG-gPLL1
was more
stable than
MPEG-gPLL2,
thus chosen for
the further
studies.

[99mTc]-pertechnetate

Fig. S3

Superose 6 10/300 GL HPLC size-exclusion column eluted with


20% acetonitrile in 0.1 M TrisHCl, pH 8.0 (0.6 mL/min).

STABILITY OF AUNP3 PROTECTIVE LAYER: SIZEEXCLUSION HPLC PROFILES OF 99mTc-AUNP3


ONE PEAK FOR
99mTc-MPEG-gPLL1
and 99mTc-AuNP3?

No [AuNP3] in
plasma

[99mTc]-pertechnetate

<4%

75% mouse

<8%

Fig. 3

AuNPS make
99mTc-MPEG-gPLL1
much more stable?

Superose 6 10/300 GL HPLC size-exclusion column eluted with


20% acetonitrile in 0.1 M TrisHCl, pH 8.0 (0.6 mL/min).

CELL UPTAKE OF AuNP3S AND CYTOTOXICITY


0.74 0.21%
10100 g Au/mL

0.70 0.31%

What
cells?

0.19 0.04%

Figure 4. (A) TEM results showing the presence of individual nonaggregated AuNP3s in the
endosomes (arrowhead). (B) Fusion of endosomes (arrowhead) with the lysosome (arrow) coinciding
with microaggregation of AuNPs. (C) Gold concentration-dependent cytotoxicity measured in cell
culture of normal human endothelial cells (black), HeLa cells (blue), and PANC-1 cells (red). (D)
Uptake of AuNP3 and control MPEG-thiol stabilized AuNP4 in HeLa, PANC-1, and normal human
endothelial cells expressed as the amount of gold taken up per million cells in culture within 24 h.
The uptake of AuNP3s was signicantly dierent between all three cell lines.

IN VIVO IMAGING SHOWING BIODISTRIBUTION


AND BIOKINETICS OF 99mTc-AuNP3
DBA/2 mouse model of
locally induced
inflammation (LPS
injection 16h before iv)
in the right femoral
muscle

0.6mCi of 99mTc-AuNP3
injected iv

Fig. 5

KINETICS OF AUNP3 ELIMINATION FROM BLOOD

8994% of 99mTc
radioactivity in the blood
was associated with
plasma and 611% with
High red blood cells and
leucocytes.
Low

Fig. 6

Blood half-lives determined from a


monoexponential t of 99mTc- AuNP3
radioactivity elimination

STABILITY OF 99mTC-AUNP3 IN VIVO

10 mg Au/kg

0.25 mg Au/kg

Fig. 6S

Superose 6 10/300 GL HPLC size-exclusion column eluted with


20% acetonitrile in 0.1 M TrisHCl, pH 8.0 (0.6 mL/min).

AUNP3 BIODISTRIBUTION

22 hours pi

20 % lower

radioactivity

5 mg Au/kg

%ID Au/organ 25 times higher


than %ID 99mTc/organ

Only account for ~50%


ID Au, ~30% ID 99mTc
Fig. 6

BIODISTRIBUTION OF 99mTcMAG3-AUNP3 IN MOUSE MODELS OF


ECTOPIC PANCREATIC CANCER AT 26H POST INJECTION

% ID 99mTc/g

Table 1S

% ID 99mTc/organ

RF HEATING AND ELECTRICAL PROPERTIES OF


AuNP3S COMPARED TO CITRATE-CAPPED AUNPS
0.75 mg gold/mL
(10.4 nm)

loss
tangent

13.56 MHz
RF operating
frequency

Fig.7

The electrical permittivity properties of the samples were


examined using a permittivity analyzer across the frequency
range 10 MHz to 1 GHz.

CONCLUSION

Coating of AuNPs with a layer of biocompatible graft


copolymer during synthesis in the water phase
resulted in nanosized single core nanoparticles
exhibiting a highly improved stability in solutions
containing physiological anions.
The improved stability in biological uids and, as
a consequence, long circulation times in vivo were
observed in the case of AuNP3 coated with more
stable MPEG-gPLL lacking ester bonds.
The ability of AuNP3s to accumulate at the sites of
abnormal vascular permeability such as sterile
inammation in vivo while resisting aggregation
after cellular uptake makes them useful for inducing
either direct or RF heating-induced cytotoxicity in
cancer cells and suggest the substantial therapeutic
and diagnostic potential of AuNP3s.

CRITIQUE1

The purity of nanoparticles was determined using a


Superdex 200 size-exclusion HPLC column (GEHealthcare Life Sciences) eluted with 0.1 M
ammonium acetate buer, pH 7.0.

No chromatograph shown and no purity given

SIZE-EXCLUSION HPLC OF MPEG-GPLL


> 660 kDa

66 kDa

150 kDa

Fig. 1S

660 kDa

Is it
possible to
determine
the purity of
AuNP3
under this
condition?

According to TNBS assay the % of


PLL amino group modification with
MPEG chains was 13-14%

100 l, 1 mg/ml, Superdex200 column (25x 1 cm), eluted with 0.1 M


ammonium acetate, pH 7.0 (0.5 ml/min). The column has been
calibrated using thyroglobulin (1); IgG (2), BSA (3).

CRITIQUE 2

Figure 3 STABILITY OF AUNP3 PROTECTIVE


LAYER

Superose 6 10/300 GL HPLC size-exclusion


column cannot separate 99mTc-MPEG-gPLL1 and
99mTc-AuNP3
HPLC profile cannot prove whether 99mTc-MPEGgPLL1 is still attached to AuNPs

CRITIQUE 3

Fig. 5 SPECT imaging of inflammation by AuNP3

Lack of a critical control: 99mTc-MPEG-gPLL1,


which itself is a blood pool imaging agent in clinical
trial

CRITIQUE 4

Fig. 6 Blood half-lives of 99mTc- AuNP3 (9.7,12.7h)

No comparison with the half-life of 99mTc-MPEGgPLL1, which has MW>660kDa, 6-9nm size and
happens to have blood half-life of ~14.1 hours

CRITIQUE 5
Fig. 6 AuNP3 biodistribution
Only account for ~50% ID Au, ~30% ID 99mTc
Actually shows the instability of AuNP3
Table 1S Biodistribution of 99mTcMAG3-AuNP3
in mouse models of ectopic pancreatic cancer at
26h post injection also shows the instability of
AuNP3 and basically no tumor uptake,
(0.210.22) % ID/tumor

REVIEWERS DECISION
Accept
Accept

with Minor Revision

Accept

with Major Revision

Reject