Journal critique of the characterization and application of CuS@MSN based theranostic for cancer

applications
Emily Thomas1,*, Hugo Pontes1,*
1
Department of Chemical Engineering, University of Washington, Seattle WA
*Authors contributed equally

Purpose and Context

In Vivo Tumor Vasculature Targeting of CuS@MSN Based Theranostic Nanomedicine introduces a theranostic
nanoparticle developed at the University of Wisconsin. Feng Chen, Hao Hong, et al. present their nanoparticle
platform as an improvement upon current cancer treatment methods. The theranostic nanoparticle entails a
copper sulfide (CuS) core with photothermal ablation properties in a mesoporous silica shell (MSN), whose
high loading capacity allows it to act as a delivery system for chemotherapeutics, while antibodies at the
particle’s surface facilitate active tumor targeting capabilities.  
Theranostic nanoparticles are the future of personalized cancer treatment; they combine diagnostics and
treatment with image guided therapy by allowing for simultaneous analysis and real time monitoring of drug
effects1. However, many theranostic nanoparticles use only passive tumor targeting strategies. In these, much
of the treatment never reaches its destination, resulting in high toxicity for the entire body. The new
nanoparticle (CuS@MSN) uses antibodies with high avidities for complexes commonly found in tumors and
targets them in vivo. The active targeting ensures that more of the treatment reaches the site of the tumor,
precipitously decreasing toxicity. The treatments offered by the new particle are also more effective as it
combines photothermal ablation and chemotherapy in a single, easily tracked nanoparticle.

Methods
Given the lack of nanoparticles that actively target in vivo tumors, the paper’s authors aimed to develop a
nanoparticle platform that had the capacity not only to diagnose and treat tumors, but also had high specificity
in the targeting process. To create a nanoparticle that could be tracked and use thermal properties to ablate
tumors, the researchers chose a copper sulfide (CuS) core for its near-infrared optical absorption and high
molar extinction coefficient. To make the core of the nanoparticle, they mixed 10mL of (0.85mg/mL) CuCl (aq),
2

40µL of cetyltrimethylammonium chloride (CTAC) and 40 mL of water before adding 50 µL of Na 2S and heated
the mixture to 90 oC in a water bath. After 15 minutes, CuS-CTAC nanoparticles were formed. The green CuS-
CTAC nanoparticles had a hydrodynamic diameter of 18.3 ± 0.2 nm and a zeta potential of 33.4 ± 1.3 mV.
The hydrodynamic diameter and zeta potential were obtained using Dynamic Light Scattering (DLS). Then, a
CTAC surfactant was used to create a positively-charged surface that stabilized the particles and allowed for
further surface engineering. To create a nanoparticle that could be loaded with a hydrophilic anticancer drug, a
mesoporous silica nanoshell (MSN) was grown on the CuS-CTAC nanoparticle. The researchers found that
manipulating the amount of excess CTAC used in the initial step allowed control over shell thickness and pore
size. So, to synthesize CuS@MSN nanoparticle, CTAC (2g, 25 wt% solution) and TEA (20mg) were dissolved
in 20 mL of prepared CuS-CTAC water solution. 200 µL of TEOS was then added at 40 µL/min at 85-90 oC.
After cooling, nanoparticles were centrifuged and washed with water. Nanoparticles were washed in a 1wt%
NaCl solution of methanol three times to remove the CTAC surfactant. Surfactant removal was confirmed via
Fourier transform infrared spectroscopy, which lacked the C-H peak at 3000-2800 cm characteristic of CTAC.
-1

Reacting 1mL of APS with 20 mL (2mg/mL) nanoparticles for 48h and washing with ethanol to remove excess
APS added amino groups to the surface of the CuS@MSN nanoparticles. Since polyethylene glycol (PEG) is
neutrally charged and biocompatible, it was attached to the nanoparticles to stabilize them during circulation
and reduce the body’s immune response. To actively target the tumor in vivo, TRC105 (a chimeric IgG1
antibody that binds to CD105 on tumor neovasculature) was also engineered onto the surface of the
CuS@MSN nanoparticles. Cu radioisotope was attached to facilitate in vivo nanoparticle imaging. This
64

allowed it to image the nanoparticles using positron emission tomography (PET) and perform biodistribution
studies. The researchers did a biodistribution study with two purposes, to see the amount of toxicity in the
nanoparticles, the nanoparticle location, and if they were passing biological barriers to target the tumor.
To test their nanoparticle platform in vivo, they used a 4T1 tumor model. Using 4 to 5-week-old female BALB/c
mice, they subcutaneously injected 2 x 10 cancer cells suspended in 100 µL of a 1:1 mixture of RPMI 1640
6

and Matrigel into the front flank of the mice. Once the tumor reached 5-8 mm several in vivo experiments were
conducted using the tumor model. They began with a long-term toxicity study, wherein after intravenous
injection of 90 mg/kg CuS@MSN they monitored the fluctuation of the mouse body weight and performed an
H&E stain in tissue from the heart, liver, spleen, lung, and kidney for signs of toxicity. They proceeded with a
photothermal ablation experiment using a 980 nm laser for 15 min and measured the tumor size using a
caliper. Lastly, they performed a vasculature targeted PET imaging where they examined PET scans after
intravenous injection of nanoparticles and quantified the number of nanoparticles in the tumor vasculature.

Results
Their nanoparticle platform provides a solution for cancer treatment that is more efficacious and has higher
targeting capabilities. Initially, they were unsuccessful in synthesizing a particle that was stable using citric acid
given that resulted in a negatively charged nanoparticle. They decided to use positively charged
cetyltrimethylammonium chloride (CTAC) to stabilize the CuS nanoparticles, resulting in slightly larger,
positively charged particles which could be coated in MSN. Manipulating excess CTAC levels yielded fine
control over nanoparticle size, yield, and monodispersity. This allowed for the creation of a nanoparticle which
combines CuS’s thermal ablation properties with an effective drug delivery system; researchers found that
CuS@MSN had twice the surface area and three times the pore volume of similarly structured nanoparticles.
After a 24-hour incubation period up to 465.1 mg/g of doxorubicin, a cancer fighting drug, could be loaded into
the MSN shell. This allows the drug to more efficiently reach its target; since it is inside the nanoparticle it can
take advantage of the direct targeting method used by CuS@MSN.  Further testing showed that the loaded
drug released faster at lower pH; this is useful because tumor microenvironments have a lower pH than the
bloodstream.
The MSN shell kept water soluble CuS nanoparticles from propagating before they reached the tumor site,
while surface engineering used polyethylene glycol (PEG) to increase biocompatibility and stability during
blood flow.
The MSN shell preserved CuS’s capacity for photoablation, to a degree. Testing showed that the CuS@MSN
nanoparticles absorbed 980 nm light and efficiently turned it into heat. In vitro tests revealed that the
magnitude of temperature change after exposure to a 980 nm laser depended, es the researchers expected,
on the power of the light and the concentration of CuS@MSN particles in the water. Five minutes of exposure
to light at 4M/cm2 raised temperatures in a .5mM mixture of CuS@MSN and water by 25 C.  Meanwhile, pure
o

water temperature rose by a mere 9 C when exposed to light. To test the nanoparticle’s photothermal ablation
o

properties in vivo, direct injection of CuS@MSN nanoparticles at the site of the tumor was combined with
periodic irradiation with a 980 nm laser. The dual treatment caused a precipitous increase in body temperature
(>17 C). Only minimal changes occurred in the laser and CuS@MSN only controls.  However, irradiating
o

tumors saw tissue burns and tumor shrinkage during the first four days for both CuS@MSN nanoparticles and
the laser-only control. After four days only the CuS@MSN nanoparticle+laser group saw continued tumor
shrinkage. In the combined treatment group, the tumor disappeared within the first two weeks, and did not
regrow in the 64-day testing period. Conversely, in both control groups the tumor grew rapidly after the fourth
day, and the CuS@MSN only control group never saw any tumor shrinkage. This demonstrated the efficacy of
the combined treatment. After confirming the treatments’ efficacy, the researchers tested the surface
engineered active targeting method. It proved to be effective, both in vivo and in vitro. The CuS@MSN uses
the antibody TRC105 to seek out endoglin (CD105) which is overexpressed in tumor microenvironments.
During in vitro testing, particles with the targeting system (targeted nanoparticles), more frequently connected
with CD105 positive human umbilical vein cells, than nanoparticles in the control group, which had blocked or
absent targeting systems, did. In vivo, the nanoparticles were labeled with 64Cu, then injected intravenously
into mice whose tumors expressed high levels of CD105. PET scans at 4, 15, and 24 hours revealed the
targeted nanoparticles had higher tumor uptake at all times measured.  This difference is particularly notable at
the 24-hour mark.
A long-term in vivo toxicity study revealed only minimal effects on weight, lung cellularity, and airway thickness.
Weight differences between treatment and control groups peaked at 7%, but by the time two months passed,
the difference decreased to 2%. Over the interval, the researchers observed no difference in appetite,
appearance, or activity. Ensuing examination of the mices’ heart, liver, spleen, lung, and kidney tissues saw
saw only negligible differences between the tissues and those of the healthy control mice, except in the case of
the lungs and the airway. The lungs had increased cellularity, which can cause breathing problems. Meanwhile
the airway thickened slightly. Airway thickening has been explained as a protective response against airway
narrowing and credited with airway hyperresponsiveness. It is particularly common in people who suffer from
asthma long term.  Both cases were mild, and none of the other characteristic indicators of toxicity chosen
were observed. This indicates that CuS@MSN nanoparticles are only minimally toxic to healthy mice injected
once with a high dose. The CuS@MSN nanoparticles’ structure and specific targeting methods make them
less toxic and more efficacious than previously developed nanoparticle platforms, offering the potential for
improved cancer treatment methods.

Weaknesses and Strengths

Effective active targeting promises decreased toxicity and increased efficacy, because the dual use of
photothermal ablation and cancer fighting drugs is very effective, and since more of the treatment is reaching
its objective rather than being dispersed throughout the body. This effect is particularly important in the context
of cancer treatment, which generally attacks the whole body, making it rife with unpleasant side effects.
CuS@MSN is also relatively easy to synthesize2, and its size can be modified easily by manipulating levels of
excess CTAC. Its surface is easily modifiable, allowing for antibodies to be attached that can that can target
specific receptors. CuS@MSN also offers the opportunity for continual monitoring and real time information on
the particle’s placement in and actions on the body. The study was rigorous and clear, effectively describing
particle behavior both in vivo and in vitro.
Furthermore, CuS@MSN’s creators adeptly accounted for patient physiology and the biological barriers the
nanoparticle would face in their design.
Revolutionary as it is, the nanoparticle had several issues. When testing the efficacy of the treatment, the
mouse subjects experienced tissue burns. This occurred in the laser only control group as well, so it clearly is
not an effect created by the nanoparticle. However, as an effect of the treatment, it should therefore be
considered. It would be useful to have a quantitative measure of burn severity to analyze whether the
treatment exacerbated the effects of the laser. Long term effects of CuS@MSN nanoparticle include slight
airway thickening and increased lung cellularity. These effects resulted from long term exposure to the
nanoparticle in healthy mice. On that note, an additional study on the long-term effects of the combined
treatment in mice with tumors would give a better picture of whether the treatment truly produces fewer side
effects.

Potential Implications
This paper showcased the development of a nanoparticle that not only used the physical properties of the
material for therapeutic purpose, but also accounted for the biological barriers of the body in its design through
the use of surface engineering. Current treatments for cancerous tumors do not actively target cancerous cells
and are highly toxic to the rest of the body. Past pre-clinical studies and clinical trials have shown that there is
a need in the field for actively targeting theranostic particles. Conjugating antibodies to the surface of the
nanoparticle allows the field to advance toward treatments that do not cause excess toxicity to the rest of the
body. A shell with potential for pH driven drug release, like that in CuS@MSN might further decrease toxicity
and increase drug potency; pH responsive nanoparticles are especially useful in cancer treatment because
hypoxia creates a lower pH in tumor microenvironments than in the bloodstream. The paper did not explore it
very much, but the particle’s pH sensitivity could springboard CuS@MSN nanoparticles toward brand new
applications. For example, using CuS@MSN nanoparticles might be an effective method for seeking out
tumors in which no active targeting antibody-compound matches can be found. It could also used beyond the
world of medicine. Acid rain lowers the pH of water; a nanoparticle with pH driven release mechanisms might
be used to release chemicals for water treatment or assist in cleanup methods some other way.
References
1. Renu Geetha Bai,Sivakumar Manickam, et al. Nanotechnology Applications for Tissue Engineering,
2015, 195-213.
2. Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-Targeted Drug Delivery of Tat
Peptide- Conjugated Monodisperse Mesoporous Silica Nanoparticles.J. Am. Chem. Soc. 2012, 134,
5722–5725.
3. Mol. Pharmaceutics 2010, 7, 6, 1913-1920.