Recent Progress of Multifunctional Molecular Probes for Triple-Negative Breast Cancer Theranostics

Abstract

Breast cancer (BC) poses a significant threat to women’s health, with triple-negative breast cancer (TNBC) representing one of the most challenging and aggressive subtypes due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Traditional TNBC treatments often encounter issues such as low drug efficiency, limited tumor enrichment, and substantial side effects. Therefore, it is crucial to explore novel diagnostic and treatment systems for TNBC. Multifunctional molecular probes (MMPs), which integrate target recognition as well as diagnostic and therapeutic functions, introduce advanced molecular tools for TNBC theranostics. Using an MMP system, molecular drugs can be precisely delivered to the tumor site through a targeted ligand. Real-time dynamic monitoring of drug release achieved using imaging technology allows for the evaluation of drug enrichment at the tumor site. This approach enables accurate drug release, thereby improving the therapeutic effect. Therefore, this review summarizes the recent advancements in MMPs for TNBC theranostics, encompassing the design and synthesis of MMPs as well as their applications in the field of TNBC theranostics.

Full text

Citation: Zhao, D.; Li, Z.; Ji, D.-K.; Xia,
Q. Recent Progress of Multifunctional
Molecular Probes for Triple-Negative
Breast Cancer Theranostics.
Pharmaceutics 2024,16, 803. https://
doi.org/10.3390/pharmaceutics16060803
Academic Editors: Tihomir Tomašiˇc
and Francesca Musumeci
Received: 14 May 2024
Accepted: 6 June 2024
Published: 14 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceutics
Review
Recent Progress of Multifunctional Molecular Probes for
Triple-Negative Breast Cancer Theranostics
Deyi Zhao 1,2, Zhe Li 1,2, Ding-Kun Ji 2, * and Qian Xia 2, 3, *
1School of Life Sciences, Shanghai University, Shanghai 200444, China; zhao@shu.edu.cn (D.Z.);
zhe-li@shu.edu.cn (Z.L.)
2Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine,
Shanghai 200127, China
3
Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine,
Shanghai Jiao Tong University, Shanghai 200127, China
*Correspondence: dingkunji@sjtu.edu.cn (D.-K.J.); xiaqian@renji.com (Q.X.)
Abstract: Breast cancer (BC) poses a significant threat to women’s health, with triple-negative breast
cancer (TNBC) representing one of the most challenging and aggressive subtypes due to the lack of
estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2
(HER2) expression. Traditional TNBC treatments often encounter issues such as low drug efficiency,
limited tumor enrichment, and substantial side effects. Therefore, it is crucial to explore novel
diagnostic and treatment systems for TNBC. Multifunctional molecular probes (MMPs), which
integrate target recognition as well as diagnostic and therapeutic functions, introduce advanced
molecular tools for TNBC theranostics. Using an MMP system, molecular drugs can be precisely
delivered to the tumor site through a targeted ligand. Real-time dynamic monitoring of drug release
achieved using imaging technology allows for the evaluation of drug enrichment at the tumor site.
This approach enables accurate drug release, thereby improving the therapeutic effect. Therefore,
this review summarizes the recent advancements in MMPs for TNBC theranostics, encompassing the
design and synthesis of MMPs as well as their applications in the field of TNBC theranostics.
Keywords: drug delivery; triple-negative breast cancer; molecular probes; molecular imaging;
treatment strategies
1. Introduction
Breast cancer (BC) is the most prevalent malignancy affecting women globally, con-
stituting approximately 31% of new cases. In 2023, its mortality rate was alarmingly high
at 15% [
1
]. The heterogeneity of BC is evident, leading to clinical categorization into three
primary subtypes based on the hormone receptor (ER and PR) and HER2 (ERBB2) status:
luminal ER-positive and PR-positive, further subdivided into luminal A and B; HER2-
positive; and TNBC [
2
]. Among these subtypes, TNBC accounts for 10–15% of BC cases
and carries the most unfavorable prognosis [3].
Common clinical treatments for TNBC include surgery, radiotherapy, and chemother-
apy. However, these traditional treatments have limitations for the treatment of TNBC.
Chemotherapy serves as the primary systemic medical treatment for TNBC, but TNBC
patients exhibit a less favorable outcome after chemotherapy compared with patients
with other BC subtypes [
4
,
5
]. Systemic chemotherapy, although a mainstay, often elicits
poor responses and severe side effects, and leads to multiple drug resistance [
6
,
7
]. Be-
yond chemotherapy, immunotherapy has demonstrated effectiveness in various tumors
and holds promise as a treatment strategy for TNBC. TNBC is particularly suitable for
immunotherapeutic approaches due to factors such as tumor immune infiltration, the
presence of neoantigens resulting from a mutational burden and higher genomic instability,
and elevated levels of immune markers such as programmed death-ligand 1 (PD-L1) and
Pharmaceutics 2024,16, 803. https://doi.org/10.3390/pharmaceutics16060803 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2024,16, 803 2 of 20
programmed cell death protein-1 (PD-1). These markers are closely associated with tumor
response, relapse, and overall outcomes [
8
]. However, TNBC’s high degree of heterogene-
ity, with multiple subtypes exhibiting distinct molecular profiles [
9
], poses a challenge as
targeted therapies are lacking for these specific subtypes [
10
]. Consequently, there is an
urgent need to develop effective new diagnostic and treatment methods and molecular
tools for TNBC.
Currently, the field has seen a promising shift in the diagnosis and treatment of
TNBC with the advent of new molecular diagnostic probes. MMPs that integrate target
recognition as well as diagnostic and therapeutic functions introduce advanced molecular
tools for TNBC theranostics. MMPs enable simultaneous drug delivery and visualization
of the lesion with minimal off-target toxicity [
11
]. Benefiting from the targeting groups,
MMPs can specifically bind to overexpressed receptors on the TNBC cell surface, thus
highly improving the positive drug delivery [
12
]. By combining them with new therapy
approaches such as photodynamic therapy (PDT) and photothermal therapy (PTT), MMPs
may overcome multidrug resistance to highly improve the treatment efficiency of TNBC [
13
].
The introduction of advanced imaging technologies such as magnetic resonance imaging
(MRI), fluorescence imaging, and positron emission tomography/single photon emission
computed tomography (PET/SPECT) imaging endows MMPs with superior spatiotemporal
imaging performance for TNBC diagnosis and visual precision treatment.
In this review, we summarize the recent advancements in MMPs for TNBC theranos-
tics (Figure 1). Firstly, we introduce the basic principles and design strategies of MMPs.
Subsequently, we discuss their specific applications in the early diagnosis and treatment,
pathological evaluation, and prognosis assessment of TNBC in detail. Finally, we address
the risks and challenges they encounter and provide insights into the future development
direction and clinical application prospects of MMPs. This review aims to comprehensively
summarize the application of MMPs in the diagnosis and treatment of TNBC, offering
inspiration for further research and clinical practice to advance the precise treatment of
this condition.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 3 of 22
Figure 1. Schematic illustration of diagnostic, therapeutic, and theranostic probes for TNBC.
2. Design of Multifunctional Molecular Probes
Multifunctional molecular probes integrate diverse capabilities within a single sys-
tem, encompassing specific targeting, diagnosis, and therapy. When constructing molec-
ular diagnostic probes for TNBC, it is crucial to carefully select specific targeting ligands,
suitable carriers, and appropriate imaging methods (Figure 2).
Figure 2. Schematic design of multifunctional molecular probes.
2.1. Targeting Groups for Multifunctional Molecular Probes
In the design of MMPs for TNBC, it is crucial to select the appropriate targeting
group. This involves considering the specificity, affinity, and biocompatibility of the mol-
ecule to ensure that it can accurately interact with TNBC-related molecules and exhibits
stability and safety in vivo. Common ligands include peptides, aptamers, antibodies, and
carbohydrates. They specifically bind to their receptors through ligand–receptor
Figure 1. Schematic illustration of diagnostic, therapeutic, and theranostic probes for TNBC.

Pharmaceutics 2024,16, 803 3 of 20
2. Design of Multifunctional Molecular Probes
Multifunctional molecular probes integrate diverse capabilities within a single system,
encompassing specific targeting, diagnosis, and therapy. When constructing molecular
diagnostic probes for TNBC, it is crucial to carefully select specific targeting ligands, suitable
carriers, and appropriate imaging methods (Figure 2).
Pharmaceutics 2024, 16, x FOR PEER REVIEW 3 of 22
Figure 1. Schematic illustration of diagnostic, therapeutic, and theranostic probes for TNBC.
2. Design of Multifunctional Molecular Probes
Multifunctional molecular probes integrate diverse capabilities within a single sys-
tem, encompassing specific targeting, diagnosis, and therapy. When constructing molec-
ular diagnostic probes for TNBC, it is crucial to carefully select specific targeting ligands,
suitable carriers, and appropriate imaging methods (Figure 2).
Figure 2. Schematic design of multifunctional molecular probes.
2.1. Targeting Groups for Multifunctional Molecular Probes
In the design of MMPs for TNBC, it is crucial to select the appropriate targeting
group. This involves considering the specificity, affinity, and biocompatibility of the mol-
ecule to ensure that it can accurately interact with TNBC-related molecules and exhibits
stability and safety in vivo. Common ligands include peptides, aptamers, antibodies, and
carbohydrates. They specifically bind to their receptors through ligand–receptor
Figure 2. Schematic design of multifunctional molecular probes.
2.1. Targeting Groups for Multifunctional Molecular Probes
In the design of MMPs for TNBC, it is crucial to select the appropriate targeting group.
This involves considering the specificity, affinity, and biocompatibility of the molecule to
ensure that it can accurately interact with TNBC-related molecules and exhibits stability and
safety
in vivo
. Common ligands include peptides, aptamers, antibodies, and carbohydrates.
They specifically bind to their receptors through ligand–receptor interactions to achieve
targeted effects on TNBC cells, thus playing an important role in the diagnosis, treatment,
and monitoring of TNBC [14].
Monoclonal antibody (mAb) is a burgeoning category of targeted cancer-treatment
drugs, distinguished by a high specificity, extended serum half-life, robust affinity, and
potent immune response function [
15
]. Monoclonal antibodies can achieve highly specific
targeting by accurately recognizing and binding to specific antigens or receptors on the
cell surface, thus exerting an excellent targeting ability in MMPs. Currently, several anti-
bodies have been successfully applied to the construction of MMPs, including m276 [
16
],
ICAM1 [
17
], and mAb
Nectin-4
[
18
]. A combination of atezolizumab and nab-paclitaxel
extended the progression-free survival of an intention-to-treat population and a PD-L1-
positive subgroup in patients with metastatic TNBC [
19
]. This ingenious approach, utilizing
antibodies for targeted drug delivery, augments drug enrichment at the tumor site, mitigat-
ing damage to normal cells and thereby enhancing the therapeutic efficacy. Guo et al. [
17
]
designed an antibody–drug conjugate by coupling the ICAM1 antibody with monomethyl
auristatin E (MMAE), verifying its excellent efficacy and safety as an effective antibody–
drug conjugate candidate drug for TNBC treatment. However, it is crucial to acknowledge
that antibodies carry certain limitations such as their substantial molecular weight and
immunogenicity. Addressing these shortcomings necessitates ongoing optimization and
improvement efforts [20].
Peptides have a series of advantages such as a simple structure, low synthesis cost, easy
engineering [
21
,
22
], strong binding affinity, and high stability [
23
], which can efficiently
reach the target and have great potential in the treatment of TNBC. To date, many peptides
have been successfully applied to the construction of MMPs, including TH19P01, AXT050,
RHFZD7, and PZ-128 [
24
]. For example, the TH19P01 peptide displayed high affinity to
the sortilin (SORT1) receptor, which is highly expressed in TNBC [
25
]. To treat TNBC,
AXT050, a collagen-IV-derived peptide with high affinity to tumor-associated integrin, was
incorporated into MMPs to achieve antitumor and antiangiogenic effects [
26
]. Doxorubicin
(DOX) is bound by peptide 18-4, targeting the TNBC surface-specific receptor keratin 1

Pharmaceutics 2024,16, 803 4 of 20
(k1), and thus has specific toxicity for TNBC [
27
]. Based on the fact that integrins and
NRP-1 are highly expressed in TNBC, the iRGD peptide, functioning as a tumor-homing
and penetrating peptide, exhibits properties of binding to neurociliin-1 and integrin
α
v
β
3,
along with being internalized into TNBC cells. The use of radionuclides to label the iRGD
peptide enables the
in vivo
imaging of TNBC [
28
]. However, peptides also have some
shortcomings such as being unstable and easily decomposed
in vivo
and having a short
half-life [29], Further modifications may be needed to improve their functions.
An aptamer is one type of single-stranded DNA or RNA. It possesses the remarkable
ability to fold into a three-dimensional structure, enabling it to selectively bind to specific
target molecules [
30
]. It can be screened from a random library using a technique called
systematic evolution of ligands by exponential enrichment (SELEX). Leveraging its high
specificity and molecular targeting prowess, aptamers have emerged as an ideal tool to
detect cancer surface markers and monitor treatment. To date, many aptamers have been
successfully applied to the construction of MMPs, including AS1411 [
31
–
35
], aptamer
(S1.5) [
36
], LXL 1 [
37
], CL4 aptamer [
38
], PD-L1 aptamer [
39
], sTN 145 aptamer [
40
], and
anti-EGFR aptamer [
41
]. Highly efficient and nuclease-resistant aptamers targeting platelet-
derived growth factor receptor
β
(PDGFR
β
) offer promising prospects to inhibit TNBC
growth. These aptamers not only contribute to antitumor immunity but also augment the
responsiveness of anti-PD-L1 antibodies in the context of TNBC growth and the formation
of lung metastases [
42
]. Epidermal growth factor receptor (EGFR) is overexpressed in about
60% of TNBC and is associated with a poor prognosis. When the cl4 aptamer was injected
into xenograft mice, it resulted in the inhibition of the formation of an integrin
α
v
β
3–
EGFR complex, thereby inhibiting tumor growth [
43
]. AS1411 can specifically target the
overexpressed nucleolar receptor on the surface of TNBC cells. It achieves its therapeutic
effect by competing with bcl-2 mRNA for nucleolin binding, thereby destabilizing bcl-2
in MDA-MB-231 [
44
]. Kang et al. [
35
] constructed AS1411 aptamer-modified PEG@PLGA
nanoparticles (NPs) encapsulated by perfluorohexane (PFH) and the anticancer drug DOX.
A further synergistic treatment of TNBC was achieved by the introduction of aptamers
to enhance tumor-targeted imaging and improve drug target enrichment. Notably, their
status as a pure nucleic-acid chain renders them biocompatible and non-immunogenic;
however, their vulnerability to nuclease degradation is an aspect worth considering [
45
,
46
].
Carbohydrates, constituting the third class of informational biomolecules following
proteins and nucleic acids, play pivotal roles in both physiological and pathological events.
These encompass critical functions in cell signaling, differentiation, proliferation, tumor
metastasis, inflammatory response, and viral infection [
47
]. Notably, carbohydrates excel
at achieving the precise targeting of cells by specifically binding to sugar receptors on the
surface of tumor cells. To date, many sugar ligands have been developed for the construc-
tion of MMPs, including hyaluronic acid (HA) [
48
–
51
] and chitosan oligosaccharide [
52
,
53
].
HA is a naturally occurring glycosaminoglycan found in the body’s connective tissue. It
can target the CD44 receptor, an integral membrane glycoprotein overexpressed on several
tumor-cell surfaces, including TNBC. Chitosan oligosaccharide can also target CD44 to
deliver MMPs to TNBC cancer cells. Dong et al. [
54
] modified liposomes using HA. This
modification helped to deliver DOX and epoprostenol (EPS) to tumor cells
in vivo
via
active targeting, which together inhibited tumor growth and metastasis in TNBC. Ding
et al. [
52
] designed a chitosan-encapsulated liposome to encapsulate a photosensitizer
(HPPH) and a hypoxia-activated prodrug (TH302) into a hydrophobic bilayer. The modi-
fication of chitosan allowed for better tumor targeting and the resulting liposome could
be used to aid the diagnosis of TNBC by fluorescent imaging and generate antitumor
therapy through synergistic PDT and chemotherapy. Leveraging these sugar molecules,
drugs, or fluorescent compounds can be conjugated to prepare MMPs, capitalizing on the
sugar transport mechanisms inherent in TNBC cells. This innovative strategy enhances
drug targeting, minimizes toxicity to normal cells, and presents a promising avenue for
therapeutic advancement.

Pharmaceutics 2024,16, 803 5 of 20
2.2. Carriers of Multifunctional Molecular Probes
Nanocarriers play a pivotal role in the construction of MMPs. Advanced nanocarriers
for MMP construction have been explored, including liposomes, hydrogels, micelles, den-
drimers, polymer NPs, and DNA nanostructures [
14
]. These carriers not only shield drugs
from degradation during transport but also amplify drug enrichment at the disease site.
Furthermore, they allow control over pharmacokinetic and pharmacodynamic distribution,
thereby mitigating adverse reactions [55].
Organic nanocarriers, including NPs such as liposomes, micelles, dendritic polymers,
and polymer nanocarriers, offer a less toxic and versatile platform for MMP construction.
These nanocarriers can be tailored to enhance drug bioavailability by improving solubility
and facilitating transport across biological membranes, thereby increasing drug absorp-
tion [
56
]. Dai et al. [
57
] targeted overexpressing integrin-
α
3 in TNBC models with cyclic
octapeptide LXY (Cys-Asp-Gly-Phe (3,5-DiF)-Gly-Hyp-Asn-Cys) attached to liposomes
carrying a dual drug, i.e., DOX and rapamycin. This dual drug targeted approach resulted
in improved efficacy compared with a free drug. Poly (acrylic acid)-g-PEG, i.e., PAA-g-
PEG, copolymeric micelles carrying DOX were developed by Sun et al. [
58
] for an efficient
reduction in lung metastasis and 4T1 mouse breast-tumor growth. The effective delivery of
therapeutic agents such as DOX, paclitaxel, and miRNA to the tumor site with the help of
nanocarriers achieves an efficient loading and release of the drugs as well as increasing the
enrichment and utilization of the drugs at the lesion site [59].
Inorganic nanocarriers, including materials such as gold, magnetic nanocarriers, quan-
tum dots, and mesoporous silica, have been utilized in the construction of MMPs. Among
these inorganic nanocarriers, mesoporous silica nanoparticles (MSNs) stand out as a novel
drug-carrier platform with advantageous biological properties, including biocompatibility,
biodegradability, and non-toxicity. Additionally, MSNs feature distinct structural attributes
such as tailored mesoporous channels, a uniform pore-size distribution, and a high surface
area, enhancing their effectiveness as drug-delivery systems [
60
,
61
]. He et al. [
62
] designed
a silica nanosystem (SNS) with Nano-Ag as the core and a complex of MnO
2
and DOX
as the surrounding mesoporous silica shell. This system was coated with anti-PD-L1 to
target the PD-L1 receptor, which is highly expressed on the surface of tumor cells. An SNS
appears to have favorable biosafety and antitumor effects, and may be a novel therapy for
the treatment of TNBC.
2.3. Imaging Modalities for Multifunctional Molecular Probes
As the prognosis of TNBC is worse than other BC subtypes, early detection, diagnosis,
and treatment are essential methods to improve its prognosis. A diagnosis of TBNC
primarily relies on morphological changes to reflect lesion characteristics. Many imaging
technologies have been successfully developed, including X-ray photography, ultrasound,
CT, MRI, and PET-CT imaging techniques [
63
]. The introduction of imaging groups to
MMPs endows them with the ability to detect TNBC. Additionally, as integral components
of molecular diagnostic probes, imaging techniques can guide therapeutic MMPs to tumors
or facilitate external triggers to induce cargo release, achieving a more precise treatment
for TNBC.
MRI is a commonly used medical imaging technology due to its high resolution and
non-invasiveness. Many MRI molecules have been explored to construct MMPs, including
As/Mn-NHs [
64
] and PDA-DNA-DTPA/Gd [
65
]. Through the labeling of drugs with
magnetic NPs or MRI contrast agents, the distribution of drugs becomes observable in
MRI, enabling a localization and distribution assessment of the drugs. Zhai et al. [
64
]
investigated self-activated As/Mn-NHs by co-biomineralizing arsenic manganite within
hollow albumin nanocages. This innovative approach demonstrated smart high-contrast
MRI detection and synergistic arseno-therapeutic effects on TNBC tumor models.
Radionuclides have gained increasing importance in modern medicine, playing a
crucial role in both the imaging and treatment of primary and metastatic tumors. Based on
their functions, they are typically categorized into two types. Diagnostic radionuclides are

Pharmaceutics 2024,16, 803 6 of 20
primarily used for tracer diagnosis in patients and include isotopes such as
99m
Tc,
68
Ga,
18
F, and
13
N [
66
]. On the other hand, therapeutic radionuclides necessitate substantial
accumulation in specific organs and rapid blood clearance. The accumulation in specific
organs should be significantly higher than that in normal tissues. Examples of therapeutic
radionuclides include
131
I,
177
Lu, and
32
P [
67
]. To date, many radioactive molecules have
been explored to construct MMPs, including
99m
Tc-HYNIC-iRGD [
28
],
99m
Tc-HYNIC-
mAb
Nectin-4
[
18
], and
89
Zr@CS-GA-MLPs [
68
]. Huang et al. [
69
] synthesized diZD, a high-
affinity vascular endothelial growth factor receptor (VEGFR)-targeting agent. This agent
was further labeled with
177
Lu and
64
Cu using a 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) chelating agent, resulting in the therapeutic agent
177
Lu-DOTA-
diZD and PET imaging agent
64
Cu-DOTA-diZD. Micro-PET/CT imaging demonstrated
a specific accumulation of
64
Cu-DOTA-diZD in 4T1 mouse tumors, while therapeutic
177Lu-DOTA-diZD exhibited remarkable antitumor efficacy.
Optical imaging uses light and the special properties of photons to obtain detailed
images of organs, tissues, cells, and even molecules. In comparison to other molecular
imaging modalities such as MRI, ultrasound imaging, and PET-CT, it boasts high spatiotem-
poral resolution and sensitivity with minimal invasiveness [
70
,
71
]. Near-infrared (NIR)
fluorophores in particular exhibit enhanced optical properties compared with traditional
fluorescent dyes. Operating within a specific window (
λ
= 700–1000 nm), NIR dyes of-
fer improved utility owing to the deeper penetration depth of the excitation light, high
spatial precision of detection, and minimized background interference of the emission
signal [
72
,
73
]. Many NIR fluorescent molecules have been explored to construct MMPs,
including Cy7 [
38
] and IR780 [
74
]. Zhang et al. [
74
] established a biomimetic nanoplatform
using a hybrid membrane derived from white blood cells and platelets (LPHMs) com-
bined with dendritic macroporous mesoporous silica nanoparticles (DLMSNs). Within the
macropores of the DLMSNs, the NIR fluorescent dye IR780 and the chemotherapeutic drug
DOX were co-loaded, resulting in DLMSN@DOX/IR780 (DDI) NPs. The biodistribution
and tumor accumulation of NPs were evaluated by observing the fluorescence of IR780.
Upon NIR laser irradiation, the LPHM@DDI NPs exhibited synergistic cytotoxicity and
apoptosis-inducing activity in TNBC cells.
3. Application of Multifunctional Molecular Probes in TNBC
TNBC, as one of the most aggressive and worst prognostic subtypes of breast cancer,
faces a number of diagnostic and therapeutic challenges due to a lack of surface receptors.
With the advancement of nanotechnology, biomedical science is increasingly focused on
the development of contrast agents and drug-delivery carriers for a more accurate and
targeted co-delivery of diagnostic and therapeutic drugs in cancer treatment [
14
]. MMPs
have been designed to encapsulate drugs within nanocarriers, linking specific ligands and
imaging moieties for the diagnosis and treatment of TNBC (Table 1).
Table 1. Summary of composition and application of MMPs.
MMPs Targeting
Group Carriers Imaging
Group Drug Application Cancer Type Mouse Strain Refs
AS-CD82-DOX-HVs AS1411 HVs DOX/CD82 MDA-MB-231 Balb/C-nude [34]
TPZ@LXL-1-PpIX-
MMT-2 LXL-1 MMT-2 PpIX MDA-MB-231 Balb/C-nude [37]
Cis-Pt@PNPs-CL4 CL4 PNPs Cy7 Cisplatin NIR MDA-MB-231/
BT-549 Balb/C-nude [38]
Cisplatin/BP/
PDA-HA HA BP Cisplatin 4T1 Balb/C [50]
As/Mn-NHs EPR Albumin Mn2+ Arsenic
trioxide (ATO) MRI 4T1/CT26/HT29 Balb/C [64]
PDA-DNA-
DTPA/Gd EPR PDA DTPA/Gd PDA/DOX MRI 4T1 Balb/C-nude [65]
177Lu-DOTA-diZD/
64Cu-DOTA-diZD diZD 64Cu 177 Lu PET 4T1 Balb/C [69]

Pharmaceutics 2024,16, 803 7 of 20
Table 1. Cont.
MMPs Targeting
Group Carriers Imaging
Group Drug Application Cancer Type Mouse Strain Refs
LPHM@DDI NPs LPHM DLMSN IR780 DOX NIR 4T1 Balb/C [74]
NaGdF4:Yb, Er
nanocrystals, EPR NaYF4
nanocrystals Gd3+ MRI/NIR LS180 Balb/C-nude [75]
cancer-cell membrane
mimic Gd3+-doped
upconversion
nanoparticles
(CCm-UCNPs)
Homologous
targeting
Cancer-cell
membrane UCNPs
UCL/MRI/PET
MDA-MB-
231/MCF-7 Balb/C-nude [76]
99mTc-HYNIC-
mAbNectin-4/
mAbNectin-4-ICG
mAbNectin-4 99mTc/ICG SPECT/NIR MDA-MB-468 Balb/C-nude [18]
[89Zr]ZrDFO-
Amivantamab Amivantamab 89Zr PET
MDA-MB-468/
MDA-MB-231/
MDA-MB-453
Balb/C-nude [77]
O2-PPSiI RGD/uPA PLGA ICG/
Gd-DTPA PTX NIR/MRI MDA-MB-231 Balb/C-nude [78]
CCm-HSA-ICG-
PFTBA
Homologous
targeting
Cancer-cell
membrane ICG/18FPFTBA NIR/PET 4T1 Balb/C-nude [79]
PepSQ@USPIO
Cys-Arg-Glu-
Lys-Ala
(CREKA)
USPIO SQ/USPIO SQ NIR/MRI MDA-MB-231/
MCF-7 Balb/C-nude [80]
[89Zr]DFO-CR011 CR011 89 Zr PET
MDA-MB-157/
MDA-MB-468/
MDA-MB-231
Balb/C-nude [81]
177Lu-NM600/
86Y-NM600 NM600 NM600 86 Y177Lu PET 4T1/4T07 Balb/C-nude [82]
[68Ga]-NOTA-GZP GZP 68Ga PTX/anti-PD-
1/anti-CTLA4 PET 4T1/E0771
Balb/C/C57Bl6
[83]
11C-methoxy-
OTSSP167 OTSSP167 11CPET MCF-7/
MDA-MB-231 Balb/C-nude [84]
3.1. MMPs for the Targeted Diagnosis of TNBC
Currently, challenges such as inaccurate diagnosis using non-specific contrast agents,
false-positive results, and the influence of examiner experience remain limiting and decisive
factors in validating TNBC diagnoses [
14
]. The evolving field of nanotechnology offers
promise, particularly through the development of nanocarrier-based molecular diagnostic
probes, which have the potential to address and overcome these limitations.
To increase the sensitivity of detection, some radionuclide-based MMPs have been de-
veloped with high affinity to TNBCs. Recent studies have highlighted nectin cell adhesion
molecule 4 (Nectin-4), also known as poliomyelitis virus receptor-related protein 4, which is
expressed in 62% of TNBC cases and is associated with a poor prognosis [
85
]. In the study
by Shao et al. [
18
], the authors utilized a mAb against Nectin-4 as a carrier to design a radio
probe,
99m
Tc-HYNIC-mAb
Nectin-4
. This radio probe exhibited significant Nectin-4-specific
targeting both
in vitro
and
in vivo
. Micro-SPECT/CT images revealed that tumor radioac-
tivity in the experimental group could be identified after 3 h, gradually increasing over
time. Notably, significant radioactivity was observed at the tumor site, with a favorable
target-background contrast at 24 and 36 h. In the study by Cavaliere et al. [
77
], a radio-
labeled bispecific antibody called [
89
Zr]ZrDFO-Amivantamab was successfully prepared
and evaluated for its pharmacological and imaging properties. The researchers compared
the imaging quality of [
89
Zr]ZrDFO-Amivantamab with a radiolabeled antibody iso-type
control, [
89
Zr]ZrDFO-IgG1. They evaluated the performance of both antibodies in terms of
their pharmacological and imaging properties. PET imaging demonstrated an increased
tumor accumulation of [
89
Zr]ZrDFO-Amivantamab in MDA-MB-468 and MDA-MB-231
compared with [89Zr]ZrDFO-IgG1, resulting in an excellent imaging contrast.
Multimodal imaging or multiplexed imaging refers to the simultaneous production
of signals for more than one imaging technique. Each imaging modality has its inherent
advantages and disadvantages. Therefore, in order to obtain multidimensional biological in-

Pharmaceutics 2024,16, 803 8 of 20
formation, different imaging modalities can be rationally combined to realize ultrasensitive
in vivo
multimodal precise imaging of TNBCs. MMPs with various imaging capabilities
show great application potential in the targeted diagnosis of TNBC. Upconversion nanopar-
ticles (UCNPs) stand out as superior luminescent substances. This superiority arises from
the facile combination of rare-earth ions with paramagnetic ions such as Gd
3+
, enabling the
simultaneous visualization of tumors through upconversion luminescence (UCL) [
86
] and
MRI [
75
,
87
]. In a study by Fang et al. [
76
], the authors designed a probe using the cancer-
cell membrane of MDA-MB-231 to modify Gd
3+
-doped UCNPs NaGdF
4
:Yb,Tm@NaGdF
4
(CCm
231
-UCNPs) (Figure 3). This probe showcased homologous-targeting and immune-
escaping abilities. Harnessing the UCL of UCNPs, the paramagnetism of Gd
3+
, and click
chemistry enabled a surface modification to label
18
F. CCm-UCNPs were employed for
ultrasensitive
in vivo
UCL/MRI/PET multimodal precise imaging of TNBC. The multiple
imaging modalities used together confirmed that the tumor sites in the MDA-MB-231
group showed much higher uptake of the probe than the MCF-7 group, thus the NPs
had the efficacy to distinguish between the MDA-MB-231 and MCF-7 hormonal mouse
models
in vivo
. This may be a potential method to achieve the integration of diagnosis and
treatment as well as to monitor and evaluate therapeutic effects.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 9 of 22
ultrasensitive in vivo multimodal precise imaging of TNBCs. MMPs with various imaging
capabilities show great application potential in the targeted diagnosis of TNBC. Upcon-
version nanoparticles (UCNPs) stand out as superior luminescent substances. This supe-
riority arises from the facile combination of rare-earth ions with paramagnetic ions such
as Gd3+, enabling the simultaneous visualization of tumors through upconversion lumi-
nescence (UCL) [86] and MRI [75,87]. In a study by Fang et al. [76], the authors designed
a probe using the cancer-cell membrane of MDA-MB-231 to modify Gd3+-doped UCNPs
NaGdF4:Yb,Tm@NaGdF4 (CCm231-UCNPs) (Figure 3). This probe showcased homolo-
gous-targeting and immune-escaping abilities. Harnessing the UCL of UCNPs, the para-
magnetism of Gd3+, and click chemistry enabled a surface modification to label 18F. CCm-
UCNPs were employed for ultrasensitive in vivo UCL/MRI/PET multimodal precise im-
aging of TNBC. The multiple imaging modalities used together confirmed that the tumor
sites in the MDA-MB-231 group showed much higher uptake of the probe than the MCF-
7 group, thus the NPs had the efficacy to distinguish between the MDA-MB-231 and MCF-
7 hormonal mouse models in vivo. This may be a potential method to achieve the integra-
tion of diagnosis and treatment as well as to monitor and evaluate therapeutic effects.
Figure 3. Illustration of the cancer-cell membrane-coated Gd3+-doped upconversion nanoparticles
(CCm-UCNPs) used to differentiate between MDA-MB-231 and MCF-7 tumor-bearing mice models
by homologous-targeting multimodality imaging, including UCL, MRI, and PET. Colors/red doed
circles/white arrows indicate tumor site. Reprinted from [76].
3.2. MMPs for the Targeted Therapy of TNBC
As one of the most malignant subtypes of BC, TNBC lacks effective therapeutic strat-
egies and has a poor prognosis. Currently, the therapeutic options for TNBC are still lim-
ited to surgery, adjuvant chemotherapy, and radiotherapy [8]. MMPs provide a new di-
rection for the precision treatment of TNBC. Zhang et al. [34] generated an exosome-mi-
metic nanovesicle system integrated with CD82 overexpression, AS1411 conjugation, and
DOX delivery (Figure 4). Exosomes are an efficient drug-delivery system and CD82 is an
exosome-enriched tumor metastasis inhibitory molecule. Nucleic-acid aptamer AS1411
specifically targets TNBC cells with a high expression of nucleolin. CD82 enrichment ef-
fectively inhibits TNBC cell migration. DOX loading effectively inhibits TNBC cell
Figure 3. Illustration of the cancer-cell membrane-coated Gd
3+
-doped upconversion nanoparticles
(CCm-UCNPs) used to differentiate between MDA-MB-231 and MCF-7 tumor-bearing mice models
by homologous-targeting multimodality imaging, including UCL, MRI, and PET. Colors/red dotted
circles/white arrows indicate tumor site. Reprinted from [76].
3.2. MMPs for the Targeted Therapy of TNBC
As one of the most malignant subtypes of BC, TNBC lacks effective therapeutic
strategies and has a poor prognosis. Currently, the therapeutic options for TNBC are still
limited to surgery, adjuvant chemotherapy, and radiotherapy [
8
]. MMPs provide a new
direction for the precision treatment of TNBC. Zhang et al. [
34
] generated an exosome-
mimetic nanovesicle system integrated with CD82 overexpression, AS1411 conjugation,
and DOX delivery (Figure 4). Exosomes are an efficient drug-delivery system and CD82 is
an exosome-enriched tumor metastasis inhibitory molecule. Nucleic-acid aptamer AS1411
specifically targets TNBC cells with a high expression of nucleolin. CD82 enrichment
effectively inhibits TNBC cell migration. DOX loading effectively inhibits TNBC cell

Pharmaceutics 2024,16, 803 9 of 20
proliferation and induces apoptosis. The results showed that the MMPs significantly
inhibited liver metastasis and ameliorated the level of apoptosis in cancer cells.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 10 of 22
proliferation and induces apoptosis. The results showed that the MMPs significantly in-
hibited liver metastasis and ameliorated the level of apoptosis in cancer cells.
Figure 4. Generation of “triple-punch” strategy cell membrane-derived nanovesicles follows three
steps: 1. overexpression of the metastasis-suppressive molecule CD82 in HBE cells; 2. extraction of
cell membrane fragments and preparation of cell membrane vesicles; and 3. modification with ap-
tamers and loading of DOX into membrane vesicles. After intravenous (i.v.) administration, AS-
CD82-DOX-HVs can specifically bind to TNBC by AS1411 targeting (a), effectively delivering DOX
to induce cancer-cell apoptosis (b) and providing metastasis inhibition from CD82 overexpression
(c), which can enhance antitumor efficacy [34]. Copyright 2024, American Chemical Society.
MMPs for the controlled delivery of drugs such as cisplatin, which can be combined
with other therapeutic modalities for synergistic antitumor effects and metastasis inhibi-
tion, are highly desirable for the treatment of TNBC. Li et al. [50] reported a multifunc-
tional molecular probe based on black phosphorus (BP) nanosheets, which were com-
posed of cisplatin, BP, polydopamine (PDA), and HA for the control of cisplatin delivery
and the inhibition of tumor growth. In this study, the BP nanosheets were sequentially
modified with PDA and HA. This molecular probe enabled the targeted and on-demand
delivery of cisplatin to inhibit tumorigenesis and metastasis through a synergistic chem-
otherapy/photothermal mode, providing a method for BP-based antitumor therapy.
To improve the efficacy of TNBC treatment, Zhang et al. [37] developed a novel strat-
egy combining bioreductive therapy with photodynamic PDT. Thin-shell hollow mesopo-
rous Ia3d silica NPs (named MMT-2) were functionalized with protoporphyrin IX (PpIX)
and a DNA aptamer (LXL-1) and loaded with tirapazamine (TPZ), resulting in the for-
mation of TPZ@LXL-1-PpIX-MMT-2 nanocarriers, which were designed to target the hu-
man BC cells, MDA-MB-231. The PpIX molecules were utilized to react and consume ox-
ygen at the tumor site to produce cytotoxic reactive oxygen species (ROS). Low oxygen
levels in the microenvironment activated the bioreductive prodrug TPZ into toxic radicals,
which not only scavenged hypoxic tumor cells but also enhanced the efficacy of PDT. The
results showed that this multifunctional molecular probe facilitated in vitro and in vivo
targeting and significantly reduced the tumor volume in xenograft mouse models. A his-
tological analysis also showed that this nanocarrier was effective at killing tumor cells in
hypoxic regions.
Figure 4. Generation of “triple-punch” strategy cell membrane-derived nanovesicles follows three
steps: 1. overexpression of the metastasis-suppressive molecule CD82 in HBE cells; 2. extraction
of cell membrane fragments and preparation of cell membrane vesicles; and 3. modification with
aptamers and loading of DOX into membrane vesicles. After intravenous (i.v.) administration, AS-
CD82-DOX-HVs can specifically bind to TNBC by AS1411 targeting (a), effectively delivering DOX to
induce cancer-cell apoptosis (b) and providing metastasis inhibition from CD82 overexpression (c),
which can enhance antitumor efficacy [34]. Copyright 2024, American Chemical Society.
MMPs for the controlled delivery of drugs such as cisplatin, which can be combined
with other therapeutic modalities for synergistic antitumor effects and metastasis inhibition,
are highly desirable for the treatment of TNBC. Li et al. [
50
] reported a multifunctional
molecular probe based on black phosphorus (BP) nanosheets, which were composed of cis-
platin, BP, polydopamine (PDA), and HA for the control of cisplatin delivery and the inhibi-
tion of tumor growth. In this study, the BP nanosheets were sequentially modified with PDA
and HA. This molecular probe enabled the targeted and on-demand delivery of cisplatin to
inhibit tumorigenesis and metastasis through a synergistic chemotherapy/photothermal
mode, providing a method for BP-based antitumor therapy.
To improve the efficacy of TNBC treatment, Zhang et al. [
37
] developed a novel strategy
combining bioreductive therapy with photodynamic PDT. Thin-shell hollow mesoporous
Ia3d silica NPs (named MMT-2) were functionalized with protoporphyrin IX (PpIX) and
a DNA aptamer (LXL-1) and loaded with tirapazamine (TPZ), resulting in the formation
of TPZ@LXL-1-PpIX-MMT-2 nanocarriers, which were designed to target the human BC
cells, MDA-MB-231. The PpIX molecules were utilized to react and consume oxygen at the
tumor site to produce cytotoxic reactive oxygen species (ROS). Low oxygen levels in the mi-
croenvironment activated the bioreductive prodrug TPZ into toxic radicals, which not only
scavenged hypoxic tumor cells but also enhanced the efficacy of PDT. The results showed
that this multifunctional molecular probe facilitated
in vitro
and
in vivo
targeting and
significantly reduced the tumor volume in xenograft mouse models. A histological analysis
also showed that this nanocarrier was effective at killing tumor cells in hypoxic regions.

Pharmaceutics 2024,16, 803 10 of 20
3.3. MMPs for the Theranostics of TNBC
Compared with independent treatment methods and imaging modes, therapeutic
MMPs have the advantage of integrating imaging and treatment into the same platform,
thereby achieving the synchronous diagnosis and treatment of tumors. Additionally, they
can monitor treatment effectiveness, enhancing the overall efficacy of tumor treatment.
MRI, fluorescence imaging, and PET have successfully been integrated into MMPs to detect
microscopic lesions, track drug release, and evaluate the effectiveness of TNBC treatment.
3.3.1. Theranostic Probes Based on MRI
MRI holds significant promise for advances in the visualized drug delivery of TNBC.
Through its capability to monitor drug distribution, release kinetics, treatment response,
and drug targeting, MRI enables the optimization of treatment plans and enhances overall
treatment effectiveness. Sun et al. [
65
] developed a therapeutic diagnostic MRI nanoprobe
(PDA-DNA-DTPA/Gd) for the imaging and treatment of a 4T1 tumor-bearing mouse
model. A PDA-DNA-DTPA/Gd solution was injected into the tumor site of 4T1 tumor-
bearing mice and whole-body MRI scans were conducted at various time points. An
optimal contrast of the MRI signal at the tumor site was observed after 30 min. Upon
irradiating the tumor site with an 808 nm laser, the non-irradiated tumor area remained
unchanged and the contrast of the tumor area after laser irradiation significantly decreased
over time. After 1 h, the MRI signal of the tumor site was significantly lower than that of
non-laser irradiation. An intertumoral injection revealed that the antitumor effect of laser-
irradiated PDA-DNA-DTPA/Gd-DOX
in vivo
was significantly higher than that of other
treatments, leading to an improved survival rate of mice. This study further demonstrated
the feasibility of synergistic PTT and chemotherapy, enhancing the therapeutic efficacy of
the nanoprobe.
In another study, Zhang et al. [
78
] introduced a NIR-responsive on-demand oxygen-
releasing nanoplatform (O
2
-PPSiI) guided by MRI, with the perspective that alleviating the
hypoxic microenvironment of TNBC could enhance the antitumor efficacy (Figure 5). The
nanoplatform was administered into the tail vein of mice with MDA-MB-231 orthotopic
tumors and exposed to an 808 nm NIR laser for 5 min. The antitumor activity of O
2
-PPSil
was assessed using structural and functional MRI. O
2
-PPSiI accumulation at the tumor
site peaked 8 h after the tail-vein injection. A further finding was that the synergistic
chemophototherapy induced by the improvement in tumor hypoxia under NIR laser
irradiation effectively suppressed tumor-cell proliferation. The nanosystem achieved
a precise O
2
release through the NIR-responsive rupture of silica shell. The real-time,
dynamic biodistribution of O
2
-PPSiI was quantitatively analyzed using sensitive MRI of
the tumor. This multifunctional molecular probe, which enabled the real-time monitoring of
the nanosystem delivery, enhanced the anti-TNBC efficacy of chemotherapy by alleviating
the hypoxic microenvironment while inhibiting its invasion and metastasis.
3.3.2. Theranostic Probes Based on NIR Fluorescence Imaging
In vivo
NIR fluorescence imaging is an emerging medical imaging method, with appli-
cations in both basic experimental and clinical research. Fluorescence, known for its rapid
response and high sensitivity, faces challenges such as the inherent fluorescence of tissues
and scattering, which have constrained its development. Consequently, insufficient diagno-
sis and treatment options for TNBC have prompted research into TNBC-therapy probes,
aiming to achieve imaging-guided treatment [
87
–
90
]. Fang et al.’s study (Figure 6) [
79
] fo-
cused on the development of a bionic oxygen-delivery nanoprobe, CCm-HSA-ICG-PFTBA,
designed for homologous targeting and hypoxia relief at tumor sites to enhance the efficacy
of TNBC xenotransplantation. The results from ICG fluorescence imaging revealed that
the fluorescence in the tumor tissues of the CCm-HSA-ICG-PFTBA group was the most
intense and persisted for 48 h. In contrast, the liver content was the lowest, indicating that
the cancer-cell membrane coating reduced uptake in the reticuloendothelial system (RES).
For
in vivo
PDT treatment, the tumor volume and weight of the CCm-HSA-ICG-PFTBA

Pharmaceutics 2024,16, 803 11 of 20
with NIR group were both smallest among all the groups and significantly decreased
compared with the untreated group. This suggests that this nanoprobe has the potential to
be clinically transformed into an effective oxygen-delivery agent to alleviate TNBC tumor
hypoxia while enhancing the therapeutic efficacy of PDT.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 12 of 22
Figure 5. The structural characterization of O
2
-PPSiI and in vivo antitumor therapeutic efficacy for
NIR-triggered O
2
-PPSiI. (a) The structure of O
2
-PPSiI. (b) TEM image of O
2
-PPSi. (c) The representa-
tive images for tumor volume derived from 3D-CUBE T2WI in each group 21 days after the treat-
ment. (d) IVIM-DWI-derived mapping of tumors in each group before and after the treatment. Black
circles indicate the tumor site. (G1: Saline; G2: Laser; G3: PTX; G4: O
2
-PPSiI; G5: PPSiI + Laser; G6:
O
2
-PSiI + Laser; G7: O
2
-PPSiI + Laser) Reprinted from [79].
3.3.2. Theranostic Probes Based on NIR Fluorescence Imaging
In vivo NIR fluorescence imaging is an emerging medical imaging method, with ap-
plications in both basic experimental and clinical research. Fluorescence, known for its
rapid response and high sensitivity, faces challenges such as the inherent fluorescence of
tissues and scaering, which have constrained its development. Consequently, insuffi-
cient diagnosis and treatment options for TNBC have prompted research into TNBC-ther-
apy probes, aiming to achieve imaging-guided treatment [87–90]. Fang et al.’s study (Fig-
ure 6) [79] focused on the development of a bionic oxygen-delivery nanoprobe, CCm-
HSA-ICG-PFTBA, designed for homologous targeting and hypoxia relief at tumor sites to
enhance the efficacy of TNBC xenotransplantation. The results from ICG fluorescence im-
aging revealed that the fluorescence in the tumor tissues of the CCm-HSA-ICG-PFTBA
group was the most intense and persisted for 48 h. In contrast, the liver content was the
lowest, indicating that the cancer-cell membrane coating reduced uptake in the reticulo-
endothelial system (RES). For in vivo PDT treatment, the tumor volume and weight of the
Figure 5. The structural characterization of O
2
-PPSiI and
in vivo
antitumor therapeutic efficacy
for NIR-triggered O
2
-PPSiI. (a) The structure of O
2
-PPSiI. (b) TEM image of O
2
-PPSi. (c) The
representative images for tumor volume derived from 3D-CUBE T2WI in each group 21 days after the
treatment. (d) IVIM-DWI-derived mapping of tumors in each group before and after the treatment.
Black circles indicate the tumor site. (G1: Saline; G2: Laser; G3: PTX; G4: O
2
-PPSiI; G5: PPSiI + Laser;
G6: O2-PSiI + Laser; G7: O2-PPSiI + Laser) Reprinted from [79].

Pharmaceutics 2024,16, 803 12 of 20
Pharmaceutics 2024, 16, x FOR PEER REVIEW 13 of 22
CCm-HSA-ICG-PFTBA with NIR group were both smallest among all the groups and sig-
nificantly decreased compared with the untreated group. This suggests that this nano-
probe has the potential to be clinically transformed into an effective oxygen-delivery agent
to alleviate TNBC tumor hypoxia while enhancing the therapeutic efficacy of PDT.
Figure 6. (a) Illustration of a biomimetic oxygen-delivery nanoprobe. Red arrows indicate tumor
site. (b) In vivo fluorescence images of TNBC xenografts after the injection of CCm-HSA-ICG-
PFTBA, HSA-ICG-PFTBA, HSA-ICG, and saline at different time points. Red circles indicate the
tumor site. Reprinted from [80].
Due to its unique tumor microenvironment that is distinct from other subtypes,
TNBC exhibits a higher metastasis rate and a more aggressive nature. Recognizing these
characteristics, numerous studies have focused on targeting various factors within the tu-
mor microenvironment, including tumor-shedding gangliosides, lactic acid, and oncopro-
teins [91]. Fibronectin has emerged as a promising targeted marker for TNBC. In Wang et
al.’s study (Figure 7) [80], a therapeutic nanoprobe named PepSQ@USPIO was designed
to specifically target fibronectin, activated by endogenous cathepsin B. The study utilized
magnetic resonance/near-infrared fluorescence (MR/NIRF) imaging for TNBC to guide
PDT. The fluorescence signal was transferred to the MDA-MB-231 tumor site 2 h after a
Pep-SQ@USPIO injection. The fluorescence intensity at the tumor site gradually increased
Figure 6. (a) Illustration of a biomimetic oxygen-delivery nanoprobe. Red arrows indicate tumor
site. (b)
In vivo
fluorescence images of TNBC xenografts after the injection of CCm-HSA-ICG-PFTBA,
HSA-ICG-PFTBA, HSA-ICG, and saline at different time points. Red circles indicate the tumor site.
Reprinted from [80].
Due to its unique tumor microenvironment that is distinct from other subtypes, TNBC
exhibits a higher metastasis rate and a more aggressive nature. Recognizing these charac-
teristics, numerous studies have focused on targeting various factors within the tumor mi-
croenvironment, including tumor-shedding gangliosides, lactic acid, and oncoproteins [
91
].
Fibronectin has emerged as a promising targeted marker for TNBC. In Wang et al.’s study
(Figure 7) [
80
], a therapeutic nanoprobe named PepSQ@USPIO was designed to specifi-
cally target fibronectin, activated by endogenous cathepsin B. The study utilized magnetic
resonance/near-infrared fluorescence (MR/NIRF) imaging for TNBC to guide PDT. The flu-
orescence signal was transferred to the MDA-MB-231 tumor site 2 h after a Pep-SQ@USPIO
injection. The fluorescence intensity at the tumor site gradually increased from 2 to 6 h after
the injection and robust tumor-specific fluorescence was observed at 6 h. The therapeutic
aspects were compared with the controls and a decrease in tumor volume over time was
observed in mice treated with Pep-SQ@USPIO and laser irradiation.

Pharmaceutics 2024,16, 803 13 of 20
Pharmaceutics 2024, 16, x FOR PEER REVIEW 14 of 22
from 2 to 6 h after the injection and robust tumor-specific fluorescence was observed at 6
h. The therapeutic aspects were compared with the controls and a decrease in tumor vol-
ume over time was observed in mice treated with Pep-SQ@USPIO and laser irradiation.
Figure 7. (a) Fibronectin targeting and CTSB-activatable Pep-SQ@USPIO nanoprobe for MR/fluores-
cence imaging and enhanced PDT of TNBC. (b) Photographs of MDA-MB-231 tumor-bearing mice
during PDT. Red circles indicate the tumor site. (c) Tumor-volume curves of mice. Data present as
mean ± SD, n = 5 (** p < 0.01) (d) Weight-growth curves of mice [81]. Copyright 2020, American
Chemical Society.
Benefiting from the low toxicity and safety of NPs and leveraging biodegradable
NPs, poly (lactic-co-glycolic acid) (PLGA) was approved by the Food and Drug Admin-
istration (FDA) for clinical use. Agnello et al. [38] utilized PLGA as a carrier for NPs; this
facilitated the connection of a novel EGFR aptamer, creating a system for the targeted de-
livery of cisplatin. To track the nanovectors’ biodistribution in vivo alongside tumor
Figure 7. (a) Fibronectin targeting and CTSB-activatable Pep-SQ@USPIO nanoprobe for
MR/fluorescence imaging and enhanced PDT of TNBC. (b) Photographs of MDA-MB-231 tumor-
bearing mice during PDT. Red circles indicate the tumor site. (c) Tumor-volume curves of mice. Data
present as mean
±
SD, n= 5 (** p< 0.01) (d) Weight-growth curves of mice [
81
]. Copyright 2020,
American Chemical Society.
Benefiting from the low toxicity and safety of NPs and leveraging biodegradable NPs,
poly (lactic-co-glycolic acid) (PLGA) was approved by the Food and Drug Administra-
tion (FDA) for clinical use. Agnello et al. [
38
] utilized PLGA as a carrier for NPs; this
facilitated the connection of a novel EGFR aptamer, creating a system for the targeted
delivery of cisplatin. To track the nanovectors’ biodistribution
in vivo
alongside tumor
targeting, NIR Cy7 was covalently labeled onto the urokinase plasminogen activator re-
ceptor (uPAR) surface. Confocal microscopy and an
in vivo
imaging analysis of the TNBC
xenografts demonstrated the prepared NPs’ targeting ability for EGFR-positive tumor cells.
The effective encapsulation of cisplatin in targeted nanocarriers showcased a superior

Pharmaceutics 2024,16, 803 14 of 20
killing effect on tumor cells compared with bare drugs and nanocarriers without or with
interference aptamers. The aptamer’s targeting and cisplatin’s chemotherapeutic effect
on the DNA damage of cancer cells synergistically achieved the goal of targeted therapy.
Additionally, the authors noted that this nanoparticle could overcome TNBC resistance to
EGFR inhibitors.
3.3.3. Theranostic Probes Based on PET
PET primarily employs radionuclides, wherein radioactive material generates positrons
within the body. These positrons interact with other electrons, enabling comprehensive
body scanning and imaging. The clinical significance of PET imaging has steadily increased,
especially since the approval of
18
F-2-fluoro-2-deoxy-D-glucose (
18
F-FDG). PET imaging
not only furnishes quantitative molecular-level information but also elucidates underly-
ing biological features. As a result, it finds frequent applications in tumor diagnosis, the
staging of newly diagnosed malignant tumors, restaging patients post-radiotherapy, and
monitoring treatment progress.
The heightened expression of glycoprotein non-metastatic B (gpNMB) in TNBC is
correlated with recurrence and metastasis, making it a viable target for the antibody–
drug conjugate glembatumumab vedotin (CDX-011). In a study led by Marquez-Nostra
et al. [
81
],
89
Zr-labeled glembatumumab ([
89
Zr]DFO-CR011) was employed to categorize
patients based on their response to CDX-011. The findings indicated that [
89
Zr]DFO-CR011
holds promise as a potential pretherapeutic diagnostic tool for CDX-011, specifically in
targeting gpNMB. This underscores its potential for clinical translation within the realm of
radiopharmacology.
In Hernandez et al.’s [
82
] study (Figure 8), radiation therapy utilized
177
Lu-labeled
alkylphosphocholine (NM600) complemented with PET imaging and
86
Y to monitor NM600
biodistribution. The
177
Lu-NM600 treatment was well-tolerated, except for mild cytopenia.
Notably, the high tumor uptake observed indicated the potential of
177
Lu-NM600 to inhibit
tumor growth and extend survival in patients lacking effective treatment options. Subse-
quent studies will explore the therapeutic potential of high linear energy transfer (LET)
radionuclides such as 225Ac, 227 Th, or 212Pb for metastatic TNBC.
Recently, advancements in granzyme B and downstream effector serine proteases
of cytotoxic T cells have provided valuable biomarkers to predict the effectiveness of im-
munotherapy. In a study conducted by Napier et al. [
83
], it was discovered that using
granzyme B for PET imaging ([
68
Ga]-NOTA-GZP) could offer early insights into immune
checkpoint blockades in TNBC following chemotherapy. To the best of the authors’ knowl-
edge, this was the first study utilizing [
68
Ga]-NOTA-GZP to evaluate the impact of immune
checkpoint inhibition combined with chemotherapy on effector cell activation in a syn-
geneic orthotopic mouse TNBC model. The findings revealed that GZP-PET could offer
quantitative information regarding effector cell activation in two types of TNBC tumor and
also monitor the tumor volume. Furthermore, it could accurately differentiate the tumor
response to treatment.
Maternal embryonic leucine zipper kinase (MELK) assumes a crucial role in regulating
tumor-cell growth, particularly in TNBC where it is abundant, and has emerged as a
promising target for molecular imaging and treatment. In the study led by Tang et al. [
84
], a
high-affinity MELK inhibitor (OTSSP167) was employed for PET imaging and a molecular
probe,
11
C-methoxy-OTSSP167, was synthesized and evaluated for its application in TNBC
PET imaging. The PET imaging results substantiated that the
11
C-methoxy-OTSSP167
molecular probe could effectively distinguish between high and low MELK expressions.
The study also anticipated future clinical use by proposing a shift to
18
F due to its longer
half-life compared with
11
C. Additionally, the use of hydrophilic and non-toxic polyethylene
glycol (PEG) was suggested to enhance water solubility, improve
in vivo
pharmacokinetics,
and reduce liver absorption.

Pharmaceutics 2024,16, 803 15 of 20
Pharmaceutics 2024, 16, x FOR PEER REVIEW 16 of 22
Figure 8. (a) Schematic representation of
177
Lu radiolabeling of NM600. (b)
86
Y-NM600 PET/CT im-
aging in murine models of TNBC. Yellow arrows indicate tumor site. (c) Results of quantitative
region of interest analyses of PET imaging in BC mice [82]. Copyright 2020, Society of Nuclear Med-
icine and Molecular Imaging.
Recently, advancements in granzyme B and downstream effector serine proteases of
cytotoxic T cells have provided valuable biomarkers to predict the effectiveness of immu-
notherapy. In a study conducted by Napier et al. [83], it was discovered that using
granzyme B for PET imaging ([
68
Ga]-NOTA-GZP) could offer early insights into immune
checkpoint blockades in TNBC following chemotherapy. To the best of the authors’
knowledge, this was the first study utilizing [
68
Ga]-NOTA-GZP to evaluate the impact of
immune checkpoint inhibition combined with chemotherapy on effector cell activation in
a syngeneic orthotopic mouse TNBC model. The findings revealed that GZP-PET could
offer quantitative information regarding effector cell activation in two types of TNBC tu-
mor and also monitor the tumor volume. Furthermore, it could accurately differentiate
the tumor response to treatment.
Maternal embryonic leucine zipper kinase (MELK) assumes a crucial role in regulat-
ing tumor-cell growth, particularly in TNBC where it is abundant, and has emerged as a
promising target for molecular imaging and treatment. In the study led by Tang et al. [84],
a high-affinity MELK inhibitor (OTSSP167) was employed for PET imaging and a molec-
ular probe,
11
C-methoxy-OTSSP167, was synthesized and evaluated for its application in
TNBC PET imaging. The PET imaging results substantiated that the
11
C-methoxy-
OTSSP167 molecular probe could effectively distinguish between high and low MELK ex-
pressions. The study also anticipated future clinical use by proposing a shift to
18
F due to
its longer half-life compared with
11
C. Additionally, the use of hydrophilic and non-toxic
polyethylene glycol (PEG) was suggested to enhance water solubility, improve in vivo
pharmacokinetics, and reduce liver absorption.
4. Conclusions and Outlook
As precision medical technology advances, MMPs have emerged as a novel and ef-
fective tool for TNBC. The integration of molecular imaging technology with molecular
probes has given rise to a new class of diagnostic and therapeutic probes, presenting an
Figure 8. (a) Schematic representation of
177
Lu radiolabeling of NM600. (b)
86
Y-NM600 PET/CT
imaging in murine models of TNBC. Yellow arrows indicate tumor site. (c) Results of quantitative
region of interest analyses of PET imaging in BC mice [
82
]. Copyright 2020, Society of Nuclear
Medicine and Molecular Imaging.
4. Conclusions and Outlook
As precision medical technology advances, MMPs have emerged as a novel and
effective tool for TNBC. The integration of molecular imaging technology with molecular
probes has given rise to a new class of diagnostic and therapeutic probes, presenting
an innovative treatment strategy. This approach enables the targeted imaging of tumor
sites, allowing the real-time monitoring of drug distribution and targeting effects
in vivo
.
Consequently, it enhances the accuracy and efficacy of diagnosis, marking a significant
advance in precision medicine for TNBC.
Although MMPs have rapidly developed in recent years, they are still in their infancy.
Many challenges need to be addressed before they enter the clinic. Firstly, for now, a lack of
highly specific biomarkers is the main reason hindering probe efficacy. Therefore, finding
specific biomarkers for TNBC is crucial for the development of effective MMPs. Secondly,
the biosafety of the nanocarrier is one of the biggest obstacles in the clinical transformation
of MMPs. This issue could be resolved by the development of biodegradable or biocompat-
ible carriers. Thirdly, simple, efficient, and accurate MMP construction methods are highly
needed to increase their functionality and efficacy in TNBC.
In conclusion, MMPs exhibit vast potential in the diagnosis and treatment of TNBC.
As technology advances and research deepens, there is a strong belief that MMPs will
contribute to the formulation of more accurate and effective diagnosis and treatment
strategies for patients with TNBC.

Pharmaceutics 2024,16, 803 16 of 20
Author Contributions: Conceptualization, Q.X. and D.-K.J.; writing—original draft preparation,
D.Z. and Z.L.; writing—review and editing, D.-K.J. and Q.X.; supervision, Q.X. and D.-K.J.; project
administration, Q.X. and D.-K.J.; funding acquisition, Q.X. and D.-K.J. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the National Key Research and Development Program of
China (No. 2020YFA0909000), the National Natural Science Foundation of China (22205139), the
Natural Science Foundation of Shanghai (22ZR1437800), the Shanghai Sailing Program (20YF1424500),
the Fundamental Research Funds for the Central Universities (2020JCPT02), and the “Clinic Plus”
Outstanding Project (2023ZYA002) from Shanghai Key Laboratory for Nucleic Acid Chemistry and
Nanomedicine.
Data Availability Statement: No new data were created or analyzed in this study.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
BC Breast cancer
BP Black phosphorus
DFO Desferrioxamine
DOX Doxorubicin
EGFR Epidermal growth factor receptor
EPR Enhanced permeability and retention
EPS Epalrestat
ER Estrogen receptor
FDA Food and Drug Administration
gpNMB Glycoprotein non-metastatic B
HA Hyaluronic acid
HER2 Human epidermal growth factor receptor 2
ICAM1 Intercellular adhesion molecule-1
IHC Immunohistochemistry
LET Linear energy transfer
mAb Monoclonal antibody
MELK Maternal embryonic leucine zipper kinase
MMAE Monomethyl auristatin E
MMP Multifunctional molecular probe
MR Magnetic resonance
MRI Magnetic resonance imaging
Nectin-4 Nectin cell adhesion molecule 4
NIR Near-infrared
NIRF Near-infrared fluorescence
NPs Nanoparticles
PD-1 Programmed cell death protein-1
PD-L1 Programmed death-ligand 1
PDA Polydopamine
PDT Photodynamic therapy
PEG Polyethylene glycol
PET Positron emission tomography
PFH Perfluorohexane
PLGA Poly (lactic-co-glycolic acid)
PpIX Protoporphyrin IX
PR Progesterone receptor
PTT Photothermal therapy
PTX Paclitaxel
RES Reticuloendothelial system
ROS Reactive oxygen species
SPECT Single photon emission computed tomography
TNBC Triple-negative breast cancer
TPZ Tirapazamine

Pharmaceutics 2024,16, 803 17 of 20
UCL Upconversion luminescence
UCNPs Upconversion nanoparticles
uPA Urokinase plasminogen activator
uPAR Urokinase plasminogen activator receptor
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