Molecular Biology: Tim-3 Targeted Nanoconjugates in Leukemia Therapy

This report delves into the use of citrate-stabilized gold nanoparticles (AuNPs) conjugated with T-cell immunoglobulin and mucin domain 3 (Tim-3) for targeted drug delivery in leukemia treatment, as explored by Yasinska et al. (2018). The study highlights the potential of AuNPs, known for their anti-inflammatory properties, to selectively target malignant leukemic cells while minimizing toxicity to healthy cells. The researchers conjugated anti-Tim3 antibodies with AuNPs using glutathione (GSH) as a linker and then covered the remaining surface with rapamycin, a cytotoxic drug. Spectrophotometry confirmed the formation of the conjugate, and stability tests showed that both the antibody and rapamycin were effectively coupled to the nanoparticles. The study demonstrated that the success of mTOR inhibition in leukemic cells is dependent on the expression of Tim-3 on the cell surface, with the nano-conjugates facilitating the delivery of rapamycin into the cells via endocytosis and GSH cleavage. The report concludes that this targeted nano-carrier system enables effective drug delivery with high efficacy, contingent on the expression of the target protein, Tim-3, and suggests further in-vivo and ex-vivo studies to validate these findings. The study offers significant advancements in cancer therapy by providing a unique approach to targeted drug delivery using nanoparticles and highlights the importance of understanding nano-immune interactions for developing immune-smart cancer nanomedicine.

Running head: Molecular Biology
Molecular Biology
Name of the Student
Name of the University
Author Note

Molecular Biology
News
Leukemia is a type of bone marrow/blood cancer. It is a fatal disease that affects
haematopoiesis along with immune defence mechanism of body. Chemotherapeutic approach
is often problematic because the drugs used for the treatment of leukemia is often toxic to the
other healthy growing cells like the stem cells and thereby affecting the normal
haematopoiesis mechanism. Hence targeted delivery of the cytotoxic drugs towards the
human malignant blood cells causing leukemia is considered as one of the promising
approach for success treatment of leukemia (Vick and Mahadevan 2016). This is because, the
targeted drug delivery model will specifically target the faulty malignant cells of leukemia
via side-passing the normal proliferating haematopoietic stem cells and thereby helping to
reduce the limit of cyto-toxicity (Vick and Mahadevan 2016).
The discovery conducted by Yasinska et al. (2018), aims to analyse the efficacy of
citrate-stabilised gold nano-particles (AuNPs) in conjugation with T-cell immunoglobulin
and mucin domain 3 as a medium for drug delivery. The importance of this approach is
AUNPs have anti-inflammatory properties and are unable to affect the cells in their own.
Thus this targeted drug delivery model will help to kill the leukemic cells selectively while
reducing overall toxicity on other healthy proliferating cells (Yasinska et al. 2018).
Yasinska et al. 2018 used gold nano-particle over the human leukemia cell-line. The
binding of the cytotoxic drugs was monitored via the expression of the actin molecules (cell
surface molecules) through phosphorylation of the mTOR pathway in Western blot analysis.
Cell surface expression of Tim-3 protein (marker protein) was analysed via FACS
(fluorescence activated cell sorter). They coupled anti-Tim antibody with gold nano-particle
via employing glutathione (GSH) as a linker. Avogardo constant was taken into consideration
in order to calculate the amount of bounded antibody (1 mole of anti-Tim-3-ScAb contained

Molecular Biology
6.023x 1023 antibodies). Following the antibody mobilization, the unbounded surface of the
gold nano-particle was covered with rapamycin (cytotoxic) molecules conjugated with GSH
ester. The success of the conjugate generation was then verified via near-UV and far-UV
spectrophotometry. The structure which is responsible for the conjugate formation was found
to be beta-plated sheet of the protein structure on the basis of far-UV and SRCD spectrum.
Thus they concluded that the single chain antibody-GSH is attached with the gold
nanoparticles via co-valent bonding while retaining beta-strand configuration. They further
tested the stability of the nano-conjugates via incubating the materials at an increasing pH
and temperature levels and showed that both anti-Tim antibody and rapamycin are coupled
with the gold nano particles via GSH that contains cysteine amino acids. It has been
previously reported that numerous antibody molecules can be successfully immobilised over
one nano-particles and hence they used 1:1 ration in order to ensure that there were adequate
space on the surface of gold nano-particles to be covered by rapamycin. They used anti-Tim-
3 to conjugate with the gold nano-particles because human myeloid leukemia cell line
express Tim-3 molecules which acts as the trafficker and a receptor for galectine-9.
Galectine-9 is a protein that protects leukemic cells from host defence. Via employing FACS
they found that undifferentiated leukemic human cell line have less surface bound tim-3 and
galectin-9 in comparison to differentiated cell lines. Thus based on the results of the Western
blot then found that successful inhibition of the mTOR-dependent phosphorylation of eIF4E-
BP in human leukemic cell line is dependent on the amount of the expression of Tim-3 on the
cell surface. Anti-Tim-3 molecules which are coupled with the gold nano-particles reacted
with human leukemic cell line and thereby performing endocytosis. GSH then undergoes
lysis via membrane bound enzymes releasing rapamycin inside the target cells. Significance
of GSH cleaving is, only free rapamycin is capable of inhibiting mTOR activity inside the
leukemic cells and cleaving of GSH sets rapamycin free. Their study also elucidated that the

Molecular Biology
in order to gain a comprehensive abrogation of mTOR activity in the THP-1 cells, the
concentration of free rapamycin has be 10 micro-meter in concentration. The summarised
version of the results showed that targeted nano-carrier enables the delivery of toxic drugs
inside the leukemic cells with high level of eficacy under the action of Tim-3 expression.
However, they also concluded that successful delivery of the cytoxic drugs inside the
leukemic cells is dependent on the expression of the target protein (Tim-3). Further studies
are required to be undertaken via employing in-vivo and ex-vivo system in order to test the
efficacy of these findings (Yasinska et al. 2018).
(Source: Yasinska et al. 2018)

Molecular Biology
Views
The discovery conducted by Yasinska et al. 2018 is immensely effective in the
domain of cancer therapy because, it provides an unique exposures towards the effective
framing to targeted drug delivery to the leukemic cells via nano-particles. According to von
Roemeling et al. (2017), nano-medicine offers unique advantage towards treating human
cancers. The human body possesses specific innate responses towards the nano-particles
(NPs). This nano-response when combined with unique pathophysiological signatures under
the tumor micro-environment can severely restrict the utility of nanomedicine under
oncological settings. Furthermore, the study conducted by Yasinska et al. 2018 showed an
actual image of advancement in the domain of cancer immune-therapies, proper
understanding of the nano-immune interactions and development of immune-smart cancer
nanomedicine, which can subsequent advantage of body’s immune functions. According to
von Roemeling et al. (2017) a better understanding of acquired biological processes that
guide the fate of nano-medicine is an integral pillar towards the development of effective
individuals’ framework for curing cancer patients. The study conducted by Yasinska et al.
2018 is relevant in this domain and thus proving to be effective in the parameter of science
and technology. The study also uncovered the opportunities in the domain of cancer nano-
medicine that capitalize the increasing understanding of the tumour biology, the cell-surface
receptors of the cancer molecules and their interactions with the nano-paticles and thereby
helping to develop effective nano-therapeutic approaches for cancer patients. This is
extremely significant in the domain of science and technology because the study gave a
detailed insight about the stimulus-triggered drug release, while facilitating delivery of the
drug to the intracellular sites via targeting cell surface molecule (Shi et al. 2017).

Molecular Biology

Molecular Biology
References
Shi, J., Kantoff, P.W., Wooster, R. and Farokhzad, O.C., 2017. Cancer nanomedicine:
progress, challenges and opportunities. Nature Reviews Cancer, 17(1), p.20.
Vick, E. and Mahadevan, D., 2016. Programming the immune checkpoint to treat
hematologic malignancies. Expert opinion on investigational drugs, 25(7), pp.755-770.
von Roemeling, C., Jiang, W., Chan, C.K., Weissman, I.L. and Kim, B.Y., 2017. Breaking
down the barriers to precision cancer nanomedicine. Trends in biotechnology, 35(2), pp.159-
171.
Yasinska, I.M., Ceccone, G., Ojea-Jimenez, I., Ponti, J., Hussain, R., Siligardi, G., Berger,
S.M., Fasler-Kan, E., Bardelli, M., Varani, L. and Fiedler, W., 2018. Highly specific targeting
of human acute myeloid leukaemia cells using pharmacologically active
nanoconjugates. Nanoscale.