DM1 Loaded Ultrasmall Gold Nanoparticles Display Significant

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DM1 loaded ultra-small gold nanoparticles display significant efficacy and improved tolerability in murine models of hepatocellular carcinoma Sarah JM Hale, Richard David Perrins, Cristina Espinosa Garc#a, Alessandro Pace, Usoa Peral, Ketan R Patel, Phil Williams, Angela Robinson, Yao Ding, Gabriele Saito, Miguel Ángel-Rodriguez, Ibon Perera, Africa Barrientos, Kelly Conlon, Steve Damment, John Porter, and Tom Coulter Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00873 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Table of content graphic 89x39mm (300 x 300 DPI)

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O

O H

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DMN

NMMM

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8c9 COOH 8

O

HO O HO

/GM

Absorbance

O

S S

OH

NG5 NGM MG5

OH

MGM %MM

MTCENMMM%8

5MM

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7MM

Wavelength 8nm9

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Time 8min9

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N5MM

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O

Volume Percentage

Count

1 2 3 4 5 OH 6 HO OH O 7 OH O S 8 DMN 9 O S COOH 8 10Au DMSOOH/O %8A S 11 HO O 12 OH O 13 HO OH 14 15 MTCENMMMNN 16 17 8d9 18 N5MM 19 20 NMMM 21 22 5MM 23 24 25 M M N / % 4 5 6 Feret Diameter 8nm9 26

/MMM

HSEPEG8ECOOH

H N OH

DTT

8b9

Bioconjugate Chemistry O

A/N4nm 8mAu9

8a9

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NMM

Size 8nm9

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Particle Diameter 8nm9

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Figure 2. MTC-100038 is internalised by hepatocellular carcinoma cell lines. a) HepG2 cells were visualised by light microscopy (x20 magnification) after treatment with nanoparticles and silver enhancement staining. Silver enhancement is only seen in cells treated with GNPs (lower panel). b) Hep3B cells were visualised by TEM following treated with nanoparticles and silver enhancement staining. Only sections treated with GNPs had clear evidence of particle distribution throughout the cytoplasm (right hand panel). 177x82mm (300 x 300 DPI)


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Figure 4. MTC-100038 nanomedicine, improves tolerability and efficacy in ectopic xenograft models of HCC. NOD/SCID mice (n=10/group) received intravenous injection of either DM1 or MTC-100038 for 1 dosing cycle of 5 consecutive days (QD5). a) change in body weight and b) tumour volumes (Hep3B) measured following treatment. c) H and E staining of tumours at the end of the study. d) tumour volumes (BEL7404) measured following treatment. Arrows indicate area of significant acellularity in the MTC-100038 treated groups. Error bars represent ± S.E.M. 177x125mm (600 x 600 DPI)


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Figure 5. MTC-100038 treatment results in significant efficacy in orthotopic models of HCC and is better than the current standard of care sorafenib. Hep3B-Luc tumour cells were implanted into the left lobe of female BalbC/Nude mice. Mice (n=10/group) received intravenous injection of either DM1, MTC-100038 or vehicle control for 2 dosing cycles of 5 consecutive days (QD5), separated by 5 days). The sorafenib treatment group received oral administration of 60mg/kg daily for 21 days. Following administration of luciferin, tumour volumes were visualised by measurement in a IVIS Lumina II imaging system a) tumour growth curves, b) images of mice with tumours in situ showing significant differences in luminescence (tumour size) between treatment groups. MTC-100038 is an effect treatment agent. Error bars represent ± S.E.M. 177x90mm (600 x 600 DPI)


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DM1 Loaded Ultra-Small Gold Nanoparticles Display Significant Efficacy and Improved Tolerability in Murine Models of Hepatocellular Carcinoma

Sarah JM Hale *1, Richard D Perrins 1, Cristina Espinosa Garcίa #1, Alessandro Pace #1, Usoa Peral #2, Ketan R Patel 1, Angela Robinson 1, Phil Williams 1, Yao Ding 1, Gabriele Saito 1, Miguel Ángel Rodriguez 2, Ibon Perera 2, Africa Barrientos 2, Kelly Conlon 1, Steve Damment 1, John Porter *1, Tom Coulter 1. 1

2

Midatech Pharma Plc, 65 Park Drive, Innovation Drive, Milton, Abingdon, OX14 4RQ.

Midatech Espana, Parque Tecnológico Ibaizabal Bidea, 800 - 2ª plta, 48160, Derio, Bizakaia.

*Corresponding

#Contributed

authors

equally

KEYWORDS Ultra-small gold nanoparticles, DM1, mertansine, drug delivery, tumour efficacy, hepatocellular carcinoma, tolerability, nanomedicine.

ABSTRACT Hepatocellular carcinoma (HCC) is the sixth commonest cancer worldwide with poor prognosis and limited options for treatment. Life expectancy after diagnosis is short, the currently available treatments are not well tolerated and have limited clinical benefit. There is a clear unmet clinical


1

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need for the development of new treatments. In this study ultra-small, 2 nm gold core nanoparticles (MidaCore™) conjugated with the potent maytansine analogue DM1 (MTC-100038), were assessed as a systemic nanomedicine for the treatment of hepatocellular carcinoma. The platform improved overall tolerability of DM1, permitting ~3-fold higher levels of drug to be administered compared to free drug. Dose for dose, MTC-100038 also facilitated delivery of ~2.0-fold higher (p=0.039) levels of DM1 to the tumour compared to free DM1. MTC-100038 produced significant efficacy (tumour growth index ~102%; p=0.5). These data indicate that incorporation of DM1 onto an ultra-small gold nanoparticle (MTC-100038) improves the therapeutic window of the drug and thereby facilitated efficacy in this tumour model. At day 17 when the control group had reached a humanely permitted tumour burden (~1500 mm3) all groups were culled, and tumour tissue was harvested for histological evaluation. H and E staining of tumour tissue sections showed dose dependent reduction in overall cellularity and formation of acellular areas in the MTC-100038 treatment groups versus the DM1 or control treatment groups (Figure 4c). These data corroborate the findings of the tumour volumes and provide preliminary evidence that MTC-100038 could facilitate the destruction of tumour cells if the drug concentration delivered is sufficiently high. In the clinical setting significant heterogeneity exists between tumour types, therefore to instil confidence for any proposed clinical translation, it is desirable to demonstrate efficacy in more than one model of the same disease. Studies were therefore initiated in 6-8 week-old female NOD/SCID mice bearing subcutaneous Bel7404 tumours. Approximately 12 days post tumour inoculation, when tumours reached ~200 mm3 animals were randomised to treatment groups of DM1 (150 µg/kg; 10 µl/g I.V. QD5×2 D0-4 + D8-12), vehicle control (Saline/5% DMSO; 10 µg/g, I.V., QD5×2 D0-4 + D8-12), MTC-100038 (337.5 µg/kg and 225 µg/kg QD5; 10 µl/g I.V., QD5×2 D0-4 + D8-12). In these studies, we also included Sorafenib (60 mg/kg; 10µl/g PO (Orally) QD21) as the current standard of care for the treatment of hepatocellular carcinoma (Figure 4d). During this experiment, for animal welfare reasons, a slightly lower drug concentration of MTC-100038 was used and there were no significant adverse clinical observations in any of the treatment groups. Efficacy measurements for these studies, were conducted at day 18 post treatment which permitted comparisons across groups as all animals were still alive at this time point. Treatment with


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sorafenib at 60 mg/kg (PO, QD21) produced a significant 63.6% TGI (p=0.0001). MTC-100038 at 225 µg/kg QD5 resulted in significant (p=0.002), ‘sorafenib like’ efficacy with a TGI of 49.1% versus that of the vehicle control group. However, MTC-100038 administered systemically at 337.5 µg/kg QD5 resulted in highly significant (p=0.0001) 102.4% TGI versus vehicle control. In contrast DM1 administered at 150 µg/kg QD5 produced no significant anti-tumour activity (p=0.764; TGI -14.4%). These data indicate that in the Bel7404 murine xenograft model, MTC100038 displayed superior efficacy compared to both the current standard of care sorafenib and the maximum tolerated dose of free DM1. There is significant debate about the applicability of xenograft models to clinical translation44 although demonstration of efficacy across a variety of model types seems to be a consensus. We therefore next sought to determine if MTC-100038 would produce significant anti-tumour effects (efficacy) in an orthotopic model of hepatocellular carcinoma. These models, although significantly more challenging and invasive, should in theory more closely resemble tumour physiology in situ. Tumour cells were implanted into the left liver lobe, however, quite unexpectedly, and for reasons undetermined, it was found in small pilot studies that Hep3B-Luc cells were not well tolerated when surgically implanted into the liver of female NOD/SCID mice. Female Balbc/nude mice were therefore used for these studies and no significant clinical issues were observed following orthotopic implantation of the Hep3B-Luc cells.

Animals were

randomised 7 days post tumour implantation when the bioluminescence intensity (1.05 × 108 photons/s) of the tumours was found to have increased over two consecutive measurements, confirming that the tumours were in exponential growth phase. Dosing was commenced at 150 µg/kg DM1 (10 µl/g I.V., QD5×2 D0-4 + D8-12), saline DMSO (vehicle control) (10 µl/g IV QD5×2 D0-4 + D8-12), 60 mg/kg sorafenib (QD21, PO, 10 µl/g), MTC-100038 450 µg/kg, 337.5


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µg/kg and 225 µg/kg (QD5×2 D0-4 + D8-12). Adverse clinical effects across treatment groups were largely insignificant. Except for 1/10 animals in the sorafenib treatment group (weight loss), another 1/10 in the 450 µg/kg MTC-100038 treatment group (slight abdominal swelling, probably not treatment related) and 1/10 in the vehicle control group (general loss in body condition). At day 21, 2/10 animals in the vehicle control group reached the humane endpoint (tumour size indicated by luminescence) and were terminated. Due to this all TGI values were calculated using day 21 measurements and the bioluminescence of the treatment groups were compared to those of the control group using a Games-Howell test to obtain “p-values”. Treatment with 60 mg/kg sorafenib gave a TGI of 78.3% (p=0.046) while 150 µg/kg DM1 gave a significantly lower TGI of 16.6%, which did not reach statistical significance (p = 0.98) versus the control group. MTC100038 treatment groups dosed at 225 µg/kg, 337.5 µg/kg and 450 µg/kg gave significant TGIs of 46.7% (p = 0.04), 72.3% TGI (p = 0.06) and 98.7% (p = 0.01) respectively, indicating that MTC100038 could produce significant efficacy in this orthotopic model of HCC (Figure 5a). As it is difficult to monitor the overall clinical effects (welfare) in orthotopic modelling, it was therefore decided that all the groups with less indication of efficacy would be culled at day 24 and tumour tissues harvested. To obtain an indication of whether MTC-100038 could improve the survival of the tumour bearing animals, and as the tumour burden was still low, the 450 µg/kg treatment group was extended to day 38. During this time, no significant adverse clinical events were observed in any of the animals tested and further luminescence measurements of this group revealed that tumour growth was slow and that the tumour masses were significantly smaller than those of the vehicle control groups harvested at the earlier timepoint (Figure 5b). Tumours were subsequently used in gross histological evaluations; H and E staining revealed that vehicle control tumours were tightly packed with many tumour cell nuclei and vascular-like structures, whereas MTC-100038


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treated tumours had large haemorrhagic vessel-like structures and the overall tumour cellularity was observed visually to be less dense (data not shown), very similar to that seen in other models tested (Figure 4c). These data provide robust evidence that MTC-100038 is efficacious in several murine xenograft models of human hepatocellular carcinoma and offered significant improvement over the current standard of care sorafenib.

Figure 4. MTC-100038 nanomedicine, improves tolerability and efficacy in ectopic xenograft models of HCC. NOD/SCID mice (n=10/group) received intravenous injection of either DM1 or MTC-100038 for 1 dosing cycle of 5 consecutive days (QD5). a) change in body weight and b) tumour volumes (Hep3B) measured following treatment. c) H and E staining of tumours at the end of the study. d) tumour volumes (Bel7404) measured following treatment. Arrows indicate area of significant acellularity in the MTC-100038 treated groups. Error bars represent ± S.E.M.


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Figure 5. MTC-100038 treatment results in significant efficacy in orthotopic models of HCC and is better than the current standard of care sorafenib. Hep3B-Luc tumour cells were implanted into the left lobe of female BalbC/Nude mice. Mice (n=10/group) received intravenous injection of either DM1, MTC-100038 or vehicle control for 2 dosing cycles of 5 consecutive days (QD5), separated by 5 days). The sorafenib treatment group received oral administration of 60mg/kg daily for 21 days. Following administration of luciferin, tumour volumes were visualised by measurement in a IVIS Lumina II imaging system a) tumour growth curves, b) images of mice with tumours in situ showing significant differences in luminescence (tumour size) between treatment groups. MTC-100038 is an effect treatment agent. Error bars represent ± S.E.M MTC-100038 improves drug delivery to tumours To determine if the tolerability conferred to DM1 after attachment to an ultra-small gold core, also offered any other mechanistic benefit in terms of drug delivery to the tumour. The accumulation of drug and gold following I.V. administration (Figure 6) was measured. Firstly, murine models bearing Hep3B subcutaneous tumours were administered with either vehicle control, free DM1 or


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MTC-100038 at equivalent doses of drug (56 µg/kg QD5; Figure 6a/b). The entire tumour was harvested from each group of animals at 2, 24 or 48 hours post the last dose and weights recorded. Homogenised tissue samples were then either treated with TCEP (to release any free drug from the GNP and extract any DM1 from thiol bearing proteins) and analysed by LC-MS/MS (Figure 6a; Figure S5 and method) or subjected to ICP-MS analysis to quantitate gold (Figure 6b). As expected, DM1 was undetectable in the vehicle control group (data not shown), however at the 2 hours post injection timepoint, analysis of tumours from the MTC-100038 and DM1 treatment groups revealed 85.6 ± 11.8 ng and 39.6 ± 9.8 ng DM1 per g of tumour tissue respectively. At the 24 and 48-hour time points the amount of free DM1 was determined as 52.5 ± 6.7 ng and 58.9 ± 4.1 ng per gram of tumour tissue for MTC-100038 (which constituted ~1.5% of the total (QD5) injected dose accumulated at the 2 hour time point) and 32.3 ± 3.4 and 28.9 ± 6.5 ng per gram of tumour tissue for DM1 respectively. These data indicate that MTC-100038 conferred a ~2.0-fold increase (p=0.039) in the amount of DM1 delivered to the tumour compared to the free DM1 drug alone, across all time points tested. Gold analysis of tumours revealed an average of 346 ng of gold per g tumour tissue across the time points tested. The ratio of gold to drug are approximately as we could expect if the particle were intact and therefore corroborate that gold conjugation is driving the enhanced uptake into the tumour. These data indicate that aside from improving systemic drug tolerability, the GNP also improves drug delivery to the tumour. In a similar study design, Bel7404 subcutaneous tumours was were also measured for both DM1 and gold. Figure 6c shows that systemically administering DM1 at 150 µg/kg QD5 I.V., resulted in 90.2 ± 16.8, 72.4 ± 10.7 and 49.9 ± 2.5 ng/g DM1 in the tumour tissue (2hrs, 24hrs and 48hrs post administration respectively). However, MTC-100038 administered at 337.5 µg/kg or 225 µg/kg QD5 resulted in 287 ± 67.1, 98.1 ± 5.4 and 110 ± 3.5 or 198 ± 9.5, 144 ± 23.5 and 89.4 ± 6.5 ng/g


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tumour tissue over the same time course respectively. These data indicate that ~2% of the total injected dose of DM1, administered in the form of MTC-100038 accumulated in the tumour at the 2 hour time point. Again, the ratio of gold to drug at the 2 hour time point are approximately as expected if the particle were intact, after which we would expect the drug to cleave and be utilised by the tumour cells. These data indicate that the actual percentage of injected dose reaching the tumours is relatively low and is in line with that reported, and hotly debated by others in the literature.45 However, importantly using the nanoparticle platform facilitates enough increase in drug delivery to produce clinically beneficial anti-tumour effects. Taken together these data support the hypothesis that the ultra-small gold platform can promote tumour uptake and could be improved further by addition of moieties that enhance targeting and/or retention within the tumour.

Figure 6. Tumour Accumulation of MTC-100038 versus free DM1 after 2, 24 and 48 hours post injection. MTC-100038 or free DM1 was injected I.V. at the concentrations indicated in the graphs. a) and c) measurements of free drug (DM1) determined by LC-MS/MS analysis. b) and


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d) measures of gold determined by ICP-MS analysis. a)/b) and c)/d) derived from Hep3b and Bel7404 tumours respectively. Error bars represent ± S.E.M. MTC-100038 displays potency in a variety of primary hepatocellular carcinoma patient derived cell lines and other tumour cell types. There is no guarantee that a tumour cell line, which has undergone significant laboratory manipulations would mirror the efficacy of primary tumours cells. Therefore, to test if MTC100038 would also be effective in a variety of primary patient derived cells, 8 freshly isolated primary cell lines derived from hepatocellular carcinoma patients were selected. Biopsies were obtained with consent from patients undergoing surgery for the treatment of their condition and a single cell suspension was obtained before treatment with various concentrations of MTC-100038. All cell lines tested were found to be susceptible to MTC-100038 with IC50s ranging from ~16190 nM (Table 1), indicating that MTC-100038 has potential as a clinical agent in the treatment of primary hepatocellular carcinoma. Since MTC-100038 is a drug carrier for DM1, and DM1 has been used clinically in antibody drug conjugates (ADCs) in a number of different disease indications,46,47 the cytotoxicity of this agent was measured on variety of tumour cell lines namely; 786-0 (human renal cell adenocarcinoma), A2780 (human ovarian carcinoma), A375 (human malignant melanoma), A431 (human squamous carcinoma), A549 (human lung carcinoma), ACHN (human metastatic renal cell carcinoma), BXPC (human pancreatic adenocarcinoma) and U87MG (human glioblastoma); IC50 values were in the range of 6-50 nM (Table 1). These data indicate that MTC-100038 may be useful in the treatment of a variety of cancers as well as hepatocellular carcinoma. DM1 Cell Type

IC50 (nM)

SD (nM)

MTC-100038 n

IC50 (nM)

SD (nM)

n


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LI-03-0014 (HCC patient derived cell line)

29

-

1

16

-

1


36

-

1

190

-

1


85

-

1

69

-

1


29

-

1

120

-

1


83

-

1

47

-

1


120

-

1

77

-

1


41

2.1

2

47

51

2

LI-03-0828TM (HCC patient derived cell line)

47

-

1

53

-

1

786-O (Human renal cell carcinoma)

32

13

5

50

9.16

5

A2780 (Human carcinoma)

ovarian

1.2

0.1

2

7.6

1.48

2

A375 (Human malignant melanoma)

80

0.5

2

7.5

1.34

2

A431 (Human squamous cell carcinoma)

38

3.0

2

6.1

0.6

2


20


A549 (Human carcinoma)

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lung

9.2

0.07

2

11

0.8

2

ACHN (Human metastatic renal cell carcinoma)

6.9

2.1

2

32

2.26

2

Bel-7404 (Human hepatocellular carcinoma)

9.4

-

1

11

-

1

BXPC-3 (Human pancreatic adenocarcinoma)

4.9

0.8

2

7.8

1.48

2

Hep3B (Human hepatocellular carcinoma)

5.0

3.5

75

16

9.48

7

HepG2 (Human hepatocellular carcinoma)

3.3

1.1

13

19

11

5

U87MG (Human glioblastoma)

3.4

1.8

58

13

5.81

11

Table 1. MTC-100038 displays potent (nM) cytotoxicity in primary HCC patient derived cell lines and various tumour cell types. CONCLUSION Described herein are the pre-clinical evaluations of the nanomedicine MTC-100038; an ultra-small (2 nm core) gold nanoparticle functionalised with the potent maytansine analogue DM1. Compared to free drug, the GNP platform improved systemic tolerability and facilitated drug delivery to HCC tumours following intravenous administration. MTC-100038 resulted in significant efficacy in several in vivo models of HCC, which was superior to the of the current standard of care sorafenib. Potent in vitro activity in various HCC patient derived cell lines as well as various tumour cell


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types, gives confidence in the development of this nanomedicine; MTC-100038 as a potentially useful nanomedicine treatment for use in a variety of oncological indications, especially where there is currently an unmet clinical need. EXPERIMENTAL METHODS Nanoparticle synthesis Carboxyl-functionalized ultra-small GNPs (MidaCore™) were synthesised at a 50 mg (gold) scale via a modified Brust-Schiffrin method.38 50ml of an aqueous (deionised water) solution of 1mercapto-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid (HS-EG(8)-COOH) (8.8 mM; Iris Biotech) and di-2´-thioethyl-α-D galactopyranoside disulfide (αGalC2) (3.2 mM; Galchimia) was prepared in a cooled jacketed flask (19ºC; Syrris) equipped with overhead stirrer (PTFE pitch blade turbine impeller). Under continuous stirring, an aqueous solution of HAuCl4 (500 μl, 0.5 M; Sigma-Aldrich) was added. The pH was adjusted to 11.6 by adding 2 M NaOH solution. A freshly prepared aqueous solution of NaBH4 (5 ml of 1.0 M) was then added as quickly as possible with vigorous stirring (800 rpm). The resulting dark brown/black solution was stirred for a further 1 hour before purification by ultra-filtration using an Amicon Ultra-15 (MWCO 10 kDa; Merck) filter by centrifugation at 4820 g, followed by several repeat washings with deionised water to give the base GNP, MTC-100011. For drug loading, DM1 (WuXi Apptech) (2.36 mM) in DMSO was carefully pipetted into a glass vial and a 38% DMSO/water solution of MTC-100011 (4 g/L Au) was added and stirred for 3 h at RT. After this time, the GNP solution was transferred to an Amicon Ultra-15 filter (MWCO 10 kDa; Merck) and diluted with water to keep the DMSO content less than 20% during centrifugation. The product was purified by several centrifugations at 4820 g, during which the DMSO content was gradually reduced by changing to deionised water. Analytical Methods


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Analytical methods used for physicochemical characterisation of GNPs have been described in detail previously elsewhere.37 Briefly, UV-Vis absorption spectra were measured using a BMG Labtech SPECTROstar Nano microwell plate spectrophotometer. Dynamic light scattering (DLS) was measured in triplicate using a Zetasizer Nano ZSP (Malvern Instruments Ltd, UK), at 25˚C in distilled water. Mean volume-weighted hydrodynamic diameter of the particles was used as a measure of GNP size. GNP-bound ligands were analysed by 1H-NMR (Bruker 400MHz NMRSpectrometer) after dissolution of gold cores by treatment with KCN/KOH (0.3 M/0.1 M, respectively in dH2O). HPLC analysis of GNPs was performed using an Agilent 1260 Infinity system with an Ascentis Express Peptide C-18 octadecyl phase column (4.6 × 100 mm, 2.7 μm; Sigma) in combination with an Ascentis Express C18 octadecyl phase guard column (4.6 × 5 mm, 2.7 µm; Sigma). The gradient elution system (A: 0.1% trifluoroacetic acid in water; B: 0.1% trifluoroacetic acid in acetonitrile) was as follows with respect to B: 0 to 2 min 20%, 2 to 8 min 100%, 8-9 min 100% then re-equilibration to 20% for 1 min before the next injection. Column oven temperature was 35°C with a flow rate of 1 ml/min). Ligand shell composition and DM1 loading were analysed by releasing the ligands from GNPs by incubation in 100 mM DTT for ~3 h. GNP identity was confirmed by mass spectrometry using an Agilent 1260 high-performance liquid chromatography with an in-line Agilent 6120 ion spray single quadrupole mass spectrometer.

The same

chromatographic conditions described above were used, except for the mobile phases which both contained 0.1% formic acid as opposed to trifluoroacetic acid. Differential centrifugal sedimentation (DCS) of GNPs for sizing analysis was performed using a CPS DC24000UHR disc centrifuge (CPS Instruments, Inc., Stuart, Florida, USA). An 8% – 24% sucrose gradient was created in 11 ml water using pro-pure proteomics grade sucrose. A series of


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injections of varying sucrose concentration were injected sequentially (from high to low concentration) to generate the gradient. This was followed by dodecane (500 µl) to reduce gradient evaporation.

The gradient was allowed to stabilise and reach thermal equilibrium for

approximately 30 minutes prior to data acquisition.

Polyvinyl chloride (PVC) calibration

standards (0.237 µm, 50 µl injection volume) were analysed prior to each GNP sample (100 µl) to ensure that the instrument was operating optimally and with a high level of accuracy. Analysis was performed at 24,000 rpm with the light detector adjusted to a position suitable for the analysis of small GNPs. Particle size was calculated based on an assumed GNP density of 5.0 g/cm-3. GNP core size was measured by transmission electron microscopy (TEM) with a JEOL JEM2100F (UHR) using a single tilt sample holder and a CMOS camera (type F216 from TVIPS, driven by the TVIPS software EMMENU4). The carbon film surface covering the 400-mesh Cu grids was rendered hydrophilic by plasma treatment in a EMITECH K100X system, before dropping 0.35 µL of a 30 µg/mL gold sample solution in water onto the copper grid and letting it desiccate. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine gold concentrations in cells from uptake studies and mouse tumour tissue following biodistribution (BD) studies. All gold analysis was completed on a NexION 300X (Perkin Elmer) instrument. Cell pellets were digested in 1600 µl tetramethylammonium hydroxide (TMAH; Sigma-Aldrich) solution containing Triton X100 (Sigma-Aldrich), under agitation for approximately 30 min. Digested cells and TMAH solution (1550 µl) were transferred to a new tube followed by the addition of TMAH solution (1 ml). Internal standard solution (2450 µl of 4 ppb iridium (SigmaAldrich) in 3% (v/v) HCl/water) was added to samples and the resulting solution was measured by ICP-MS and quantitated using a calibration curve.


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Tumour tissue was weighed and transferred to a Teflon microwave vessel followed by the addition of TMAH solution containing Triton X100 (3 ml). Tissues were digested using a CEM Discover Microwave digestion system (CEM, Buckingham, UK). A small volume (350 µl) of the digested material was transferred to a fresh tube and both TMAH solution (2150 µl) and internal standard (2150 µl) were added prior to gold determination on the ICP-MS with an appropriate calibration curve. Tumour tissue and cell pellets were diluted as necessary depending on their gold content. Cells and animals Bel7404, HepG2, Hep3B and Hep3B-Luc (human hepatocellular carcinoma cell lines), 786-0 (human renal cell adenocarcinoma), A2780 (human ovarian carcinoma), A375 (human malignant melanoma), A431 (human squamous carcinoma), A549 (human lung carcinoma), ACHN (human metastatic renal cell carcinoma), BXPC (human pancreatic adenocarcinoma) and U87MG (human glioblastoma) were obtained from either ATCC or WuXi Apptec and cultured in complete DMEM (Sigma-Aldrich) supplemented with 10% (v/v) fetal calf serum (FCS; Thermo Scientific). Hep3BLuc (luciferase expressing) cells were maintained as in vitro monolayers and sub-cultured regularly by treatment with trypsin-EDTA to ensure the cells were in exponential growth phase. Patient derived cell lines were supplied and passaged by WuXi Apptec (Shanghai); primary tumour cells from consenting Hepatocellular carcinoma patients were originally established from surgically resected clinical samples, implanted into nude mice and propagated by serial transplantation. The following patient derived xenograft (PDX) tumour models were used in our studies (LI-03-257, LI-03-828TM, LI-03-141, LI-03-115, LI-03-061, LI-03-014, LI-03-143, LI03-187, LI-03-357, LI-03-309). All animal studies were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC), the Association for Assessment and Accreditation of


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Laboratory Animal Care (AAALAC) or the necessary project licences and conducted according to strict welfare protocols. Humane endpoints were used in all studies. For biodistribution studies, animals were injected with dosing solution via the tail vein (10 µl/g). Organs were harvested at the terminal endpoint, mechanically homogenised and either digested as described above prior to ICP-MS determination of gold concentrations or drug extracted and measured by LC-MS/MS, using appropriate calibration curves and internal standards (see analytical methods). Quantities are expressed as Weight/Weight. Tolerability and efficacy studies For tolerability and efficacy studies 8-14 week-old female NOD/SCID or BALB/c Nude mice, were obtained from Beijing Vital River Laboratories Co., LTD, housed in individually ventilated cages and handled using aseptic techniques. Animals were acclimatised for a suitable period prior to study assignment before drug dosing commenced. For ectopic tumours, 1×107 tumour cells were inoculated subcutaneously into the right flank in 200 µl of phosphate buffered saline (PBS)/Matrigel (1:1; Sigma-Aldrich and BD Biosciences respectively). For orthotopic tumours, mice were anesthetized, and the surgical site was prepared under sterile conditions. A small incision across the abdominal wall was made to expose the liver and ~3×106 Hep3B-Luc cells were mixed with BD Matrigel in 20 µl (PBS : Matrigel = 1:1) and then injected into the left lobe of the liver. The skin was closed with surgical sutures and the animals were continuously monitored until they had completely recovered from anaesthesia. Analgesia was administered prophylactically. For ectopic models, animals were assigned to treatment groups when the average tumour volume reached approximately 200 mm3 (~day 14 post inoculation). For orthotopic models, animals were selected and randomized (based on their average bioluminescence intensity ~1.05×108


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photons/sec) on day 7 after tumour implantation when the relative luminescence had increased for 2 consecutive measurements, indicating that the tumours were in a growth phase. Bioluminescence was monitored using an IVIS (Lumina II) imaging system. Prior to measurements, animals were anaesthetised and luciferin (150 mg/kg) was administered via intraperitoneal (I.P.) injection to facilitate tumour visualisation. Mice (10 mice/group) bearing established tumours, were then randomized to groups using a randomized block design based upon their bioluminescence or tumour volumes. For all studies dosing with the test articles (10 µl/g) then commenced daily for 5 days (QD5) for 1 or 2 cycles (separated by 5 days). For ectopic models, tumour size was measured twice weekly in two dimensions using a caliper. The volume was recorded in mm3 using the formula: V = 0.5×ab2 where a and b are the long and short diameters of the tumour, respectively. For orthotopic models, tumour bioluminescence was measured and recorded once per week. Tumour growth curves were then plotted as bioluminescence intensity (photons/sec). Tumour Growth Index (TGI) was calculated for each group using the formula: TGI (%) = [1-(TiT0)/ (Vi-V0)] ×100; Ti is the average tumour volume or bioluminescence of a treatment group on a given day, T0 is the average tumour volume/bioluminescence of the treatment group on the day of treatment start, Vi is the average tumour volume/bioluminescence of the vehicle control group on the same day with Ti, and V0 is the average tumour volume/bioluminescence of the vehicle group on the day of treatment initiation. DM1 and MTC-100038 was administered systemically for 5 consecutive days (QD5) for 1 or 2 cycles, at the doses indicated in the Figure legends. Cytotoxicity assays The patient-derived cell lines (PDX) LI-03-0257, LI-03-0828TM, LI-03-0141, LI-03-0115, LI-030061, LI-03-0014, LI-03-0143, LI-03-0187, LI-03-0357 & LI-03-0309 (passage 3-4) were generated by WuXi Apptech (Suzhou, China). Cell viability assays with Bel7404 (Cell Bank of


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the Chinese Academy of Sciences) and the PDX lines were also carried out by WuXi Apptech using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega) following the manufacturer’s protocol. Cells were plated at 2×104 cells/well into Greiner 655090 Cellstar 96well plates and incubated with a 10-point titration of DM1 or an equivalent concentration of MTC100038 (points 1-9 starting at 1 µM DM1 or equivalent GNP concentration with a 3-fold dilution between points and the final point lacked DM1 or GNP). Cells were incubated for 3-6 days, as indicated in the Figure legends, before cytotoxicity was measured.

Remaining cell lines were obtained from ATCC and expanded by SAL scientific (UK). Cells were plated at 1-2×104 cells/well into TC-treated 96-well plates (Eppendorf) and cultured overnight in L-Glutamine containing EMEM (Sigma-Aldrich) supplemented with 10% (v/v) FBS, nonessential amino acids (Sigma-Aldrich) and sodium pyruvate (Sigma-Aldrich), at 37°C & 5% CO2 atmosphere. Media was exchanged for EMEM containing compound/GNP. Unless stated cells were incubated with compound in EMEM in an 8-point 3-fold dilution series in triplicate for 3days at 37°C & 5% CO2. Cell viability was assessed with an MTT assay48 with the following alterations: cells were incubated with 0.5 mg/ml MTT (Sigma-Aldrich) in EMEM for 1 hr; formazan was solubilised in DMSO; and the absorbance was measured at 595 nm. Readouts from all cell viability assays were normalised and an IC50 obtained by fitting a four parameter [Inhibitor] vs. response curve using GraphPad Prism 7. Incubation of GNPs with cell monolayers HepG2 or Hep3B were plated overnight in either 96 well plates, Thermanox cover slips (ThermoScientific) or 6 well plates for visualisation by light microscopy, transmission electron microscopy (TEM) or analysis by ICP-MS respectively. Growth medium was replaced with freshly prepared


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solutions of test GNPs in low FBS medium (2%) at a concentration of 1.0×109 GNPs per cell (20 μl volume). Cultures were incubated for 60 min at 37°C, before being washed with warm PBS. Cells for light microscopy were then fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) solution (50 μl per well) and incubated at room temperature for 10 min. Cell cultures were permeabilised by addition of Triton X-100 in PBS (0.1%, 50 μl per well) for 10 min, then carefully washed with PBS. Working quickly, cell cultures were treated with the pre-mixed silver stain solutions at room temperature (100 μl; Sigma Aldrich) developed for 5 minutes and then washed extensively with dH20 prior to imaging by light microscopy under ×20 magnification. For TEM, cells were processed as described elsewhere49. For ICP-MS analysis, following incubation with GNPs, cells were incubated for 10 mins at room temperature with 0.2 M Acetic acid : 0.5 M NaCl pH2.8 (Sigma-Aldrich) before several washes with PBS. Cells were then harvested by scraping and subsequently analysed by ICP-MS. Statistics Data are presented as means ± standard deviation (SD) from at least 3 independent experiments, unless otherwise stated. Statistical significance was calculated using either GraphPad Prism or SPSS 17.0 software. Either a one-way ANOVA was performed when a significant F-statistic (a ratio of treatment variance to the error variance) was obtained, otherwise, Games-Howell tests were performed when homogeneity of variances was not assumed.

P values ≤ 0.05 were

considered statistically significant.

SUPPLEMENTARY INFORMATION Figure S1. Zeta potential analysis of MTC38. Figure S2.


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NMR analysis resolves peaks for Sugar and PEG ligands. Note the technique cannot be used to resolve DM1 due to peak interference. Figure S3. Cytotoxicity data (MTT assay) revealing no apparent cytotoxicity after incubation of HEP3B cells with MTC-100011 (Equivalent of MTC-100038 minus DM1). Figure S4. Relative biodistribution (%) of MTC-100011 in healthy wistar rats. (n=4 per group, harvested at 24 hours post injection of 300 µg/kg gold. Note a significant proportion of the gold is excreted into urine via kidneys. Recovered dose >95%. Figure S5. HPLC-UV Chromatograms of MTC-100038 showing release of DM1 after treatment with TCEP Quantification of DM1 loading on GNPs by DM1 release-Detailed Methodology

REFERENCES (1) (2) (3) (4) (5) (6)

(7)

(8) (9)

McGlynn, K. A., Petrick, J. L., and London, W. T. (2015) Global epidemiology of hepatocellular carcinoma: an emphasis on demographic and regional variability. Clinics in liver disease 19, 223-238. Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D. M., Forman, D., and Bray, F. (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136, E359-86. Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2001) Estimating the world cancer burden: Globocan 2000. Int J Cancer 94, 153-6. Topalian, S. L., Drake, C. G., and Pardoll, D. M. (2015) Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 27, 450-61. Zhong, J. H., Du, X. K., Xiang, B. D., and Li, L. Q. (2016) Adjuvant sorafenib in hepatocellular carcinoma: A cautionary comment of STORM trial. World J Hepatol 8, 95760. Lopus, M., Oroudjev, E., Wilson, L., Wilhelm, S., Widdison, W., Chari, R., and Jordan, M. A. (2010) Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules. Mol Cancer Ther 9, 2689-99. Poon, K. A., Flagella, K., Beyer, J., Tibbitts, J., Kaur, S., Saad, O., Yi, J. H., Girish, S., Dybdal, N., and Reynolds, T. (2013) Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability. Toxicol Appl Pharmacol 273, 298-313. Issell, B. F., and Crooke, S. T. (1978) Maytansine. Cancer Treat Rev 5, 199-207. Diamantis, N., and Banerji, U. (2016) Antibody-drug conjugates--an emerging class of cancer treatment. Br J Cancer 114, 362-7.


30


(10) (11) (12) (13) (14) (15) (16) (17)

(18) (19) (20) (21) (22)

(23) (24) (25)

Page 38 of 41

Zhao, J., Lee, P., Wallace, M. J., and Melancon, M. P. (2015) Gold Nanoparticles in Cancer Therapy: Efficacy, Biodistribution, and Toxicity. Current pharmaceutical design 21, 424051. Zhao, L., Seth, A., Wibowo, N., Zhao, C. X., Mitter, N., Yu, C., and Middelberg, A. P. (2014) Nanoparticle vaccines. Vaccine 32, 327-37. Kong, F. Y., Zhang, J. W., Li, R. F., Wang, Z. X., Wang, W. J., and Wang, W. (2017) Unique Roles of Gold Nanoparticles in Drug Delivery, Targeting and Imaging Applications. Molecules (Basel, Switzerland) 22. Jain, S., Hirst, D. G., and O'Sullivan, J. M. (2012) Gold nanoparticles as novel agents for cancer therapy. The British Journal of Radiology 85, 101-113. Dreaden, E. C., Austin, L. A., Mackey, M. A., and El-Sayed, M. A. (2012) Size matters: gold nanoparticles in targeted cancer drug delivery. Therapeutic delivery 3, 457-478. Ahmedah, H. T., Patterson, L. H., Shnyder, S. D., and Sheldrake, H. M. (2017) RGDBinding Integrins in Head and Neck Cancers. Cancers (Basel) 9. Goel, R., Shah, N., Visaria, R., Paciotti, G. F., and Bischof, J. C. (2009) Biodistribution of TNF-alpha-coated gold nanoparticles in an in vivo model system. Nanomedicine (Lond) 4, 401-10. Morshed, R. A., Muroski, M. E., Dai, Q., Wegscheid, M. L., Auffinger, B., Yu, D., Han, Y., Zhang, L., Wu, M., Cheng, Y., et al. (2016) Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer. Mol Pharm 13, 1843-54. Kumar, A., Huo, S., Zhang, X., Liu, J., Tan, A., Li, S., Jin, S., Xue, X., Zhao, Y., Ji, T., et al. (2014) Neuropilin-1-targeted gold nanoparticles enhance therapeutic efficacy of platinum(IV) drug for prostate cancer treatment. ACS Nano 8, 4205-20. Sheng, X., Huang, T., Qin, J., Li, Q., Wang, W., Deng, L., and Dong, A. (2017) Preparation, pharmacokinetics, tissue distribution and antitumor effect of sorafenibincorporating nanoparticles in vivo. Oncol Lett 14, 6163-6169. Song, K., Xin, M., Yu, H., Zheng, Z., Li, J., Li, M., Guo, H., Tan, Y., and Wu, X. (2018) Novel ultra-small micelles based on rebaudioside A: A potential nanoplatform for ocular drug delivery. Int J Pharm 552, 265-276. Sztandera, K., Gorzkiewicz, M., and Klajnert-Maculewicz, B. (2018) Gold nanoparticles in cancer treatment. Mol Pharm. Liu, R., Xiao, W., Hu, C., Xie, R., and Gao, H. (2018) Theranostic size-reducible and no donor conjugated gold nanocluster fabricated hyaluronic acid nanoparticle with optimal size for combinational treatment of breast cancer and lung metastasis. J Control Release 278, 127-139. Ruan, S., Cao, X., Cun, X., Hu, G., Zhou, Y., Zhang, Y., Lu, L., He, Q., and Gao, H. (2015) Matrix metalloproteinase-sensitive size-shrinkable nanoparticles for deep tumor penetration and pH triggered doxorubicin release. Biomaterials 60, 100-10. Ruan, S., Hu, C., Tang, X., Cun, X., Xiao, W., Shi, K., He, Q., and Gao, H. (2016) Increased Gold Nanoparticle Retention in Brain Tumors by in Situ Enzyme-Induced Aggregation. ACS Nano 10, 10086-10098. Koonce, N. A., Quick, C. M., Hardee, M. E., Jamshidi-Parsian, A., Dent, J. A., Paciotti, G. F., Nedosekin, D., Dings, R. P., and Griffin, R. J. (2015) Combination of Gold Nanoparticle-Conjugated Tumor Necrosis Factor-alpha and Radiation Therapy Results in


31

Page 39 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) (27)

(28) (29)

(30)

(31) (32)

(33) (34)

(35) (36) (37)

(38)

a Synergistic Antitumor Response in Murine Carcinoma Models. Int J Radiat Oncol Biol Phys 93, 588-96. Jiang, J., and Bischof, J. (2010) Effect of timing, dose and interstitial versus nanoparticle delivery of tumor necrosis factor alpha in combinatorial adjuvant cryosurgery treatment of ELT-3 uterine fibroid tumor. Cryo Letters 31, 50-62. Pedro, R. N., Thekke-Adiyat, T., Goel, R., Shenoi, M., Slaton, J., Schmechel, S., Bischof, J., and Anderson, J. K. (2010) Use of tumor necrosis factor-alpha-coated gold nanoparticles to enhance radiofrequency ablation in a translational model of renal tumors. Urology 76, 494-8. Visaria, R., Bischof, J. C., Loren, M., Williams, B., Ebbini, E., Paciotti, G., and Griffin, R. (2007) Nanotherapeutics for enhancing thermal therapy of cancer. Int J Hyperthermia 23, 501-11. Libutti, S. K., Paciotti, G. F., Byrnes, A. A., Alexander, H. R., Jr., Gannon, W. E., Walker, M., Seidel, G. D., Yuldasheva, N., and Tamarkin, L. (2010) Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine. Clin Cancer Res 16, 6139-49. Zarschler, K., Rocks, L., Licciardello, N., Boselli, L., Polo, E., Garcia, K. P., De Cola, L., Stephan, H., and Dawson, K. A. (2016) Ultrasmall inorganic nanoparticles: State-of-theart and perspectives for biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine 12, 1663-1701. Bhardwaj, V., and Kaushik, A. (2017) Biomedical Applications of Nanotechnology and Nanomaterials. Micromachines (Basel) 8. Huang, K., Ma, H., Liu, J., Huo, S., Kumar, A., Wei, T., Zhang, X., Jin, S., Gan, Y., Wang, P. C., et al. (2012) Size-dependent localization and penetration of ultrasmall gold nanoparticles in cancer cells, multicellular spheroids, and tumors in vivo. ACS Nano 6, 4483-93. Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X., and Zheng, J. (2013) Passive tumor targeting of renal-clearable luminescent gold nanoparticles: long tumor retention and fast normal tissue clearance. J Am Chem Soc 135, 4978-81. Amirthalingam, E., Rodrigues, M., Casal-Dujat, L., Calpena, A. C., Amabilino, D. B., Ramos-Lopez, D., and Perez-Garcia, L. (2015) Macrocyclic imidazolium-based amphiphiles for the synthesis of gold nanoparticles and delivery of anionic drugs. J Colloid Interface Sci 437, 132-139. Pooja, D., Panyaram, S., Kulhari, H., Reddy, B., Rachamalla, S. S., and Sistla, R. (2015) Natural polysaccharide functionalized gold nanoparticles as biocompatible drug delivery carrier. Int J Biol Macromol 80, 48-56. Brust, M., Walker, M., Bethell, D., Schiffrin, D. J., and Whyman, R. (1994) Synthesis of thiol-derivatised gold nanoparticles in a two-phase Liquid-Liquid system. Journal of the Chemical Society, Chemical Communications, 801-802. Roskamp, M., Coulter, T., Ding, Y., Perrins, R., Espinosa-Garcia, C., Pace, A., Hale, SJM., Robinson, A., Williams, P., Aguilera Peral, U et al., (2017) SIKVAV peptide functionalized ultra-small gold nanoparticles for selective targeting of α6β1 integrin in hepatocellular carcinoma. J.Phys.: Conf. Ser. 829 012017. Templeton, A. C., Wuelfing, W. P., and Murray, R. W. (2000) Monolayer-Protected Cluster Molecules. Accounts of Chemical Research 33, 27-36.


32


(39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49)

Page 40 of 41

Blanco, E., Shen, H., and Ferrari, M. (2015) Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol 33, 941-51. Li, B., and Lane, L. A. (2018) Probing the biological obstacles of nanomedicine with gold nanoparticles. Wiley Interdiscip Rev Nanomed Nanobiotechnol, e1542. Maeda, H., Nakamura, H., and Fang, J. (2013) The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev 65, 71-9. Hedrich, W. D., Fandy, T. E., Ashour, H. M., Wang, H., and Hassan, H. E. (2018) Antibody-Drug Conjugates: Pharmacokinetic/Pharmacodynamic Modeling, Preclinical Characterization, Clinical Studies, and Lessons Learned. Clin Pharmacokinet 57, 687-703. Mugera, G. M., and Ward, J. M. (1977) Acute toxicity of maytansine in F344 rats. Cancer Treat Rep 61, 1333-8. Lampreht Tratar, U., Horvat, S., and Cemazar, M. (2018) Transgenic Mouse Models in Cancer Research. Front Oncol 8, 268. Wilhelm, S., Tavares, A. J., Dai, Q., Ohta, S., Audet, J., Dvorak, H. F., and Chan, W. C. W. (2016) Analysis of nanoparticle delivery to tumours. Nature Reviews Materials 1, 16014. Polakis, P. (2016) Antibody Drug Conjugates for Cancer Therapy. Pharmacol Rev 68, 319. Thomas, A., Teicher, B. A., and Hassan, R. (2016) Antibody-drug conjugates for cancer therapy. Lancet Oncol 17, e254-e262. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63. Gromnicova, R., Kaya, M., Romero, I. A., Williams, P., Satchell, S., Sharrack, B., and Male, D. (2016) Transport of Gold Nanoparticles by Vascular Endothelium from Different Human Tissues. PLoS One 11, e0161610.


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DM1 Loaded Ultrasmall Gold Nanoparticles Display Significant

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