Abstract
Customizable and number‐tunable enzyme delivery nanocarriers will be
useful in tumor therapy. Herein, a phage vehicle, T4‐Lox‐DNA‐Fe (TLDF),
which adeptly modulates enzyme numbers using phage display technology
to remodel the tumor microenvironment (TME) is presented. Regarding the
demand for lactic acid in tumors, each phage is engineered to display
720 lactate oxidase (Lox), contributing to the depletion of lactic acid
to restructure the tumor's energy metabolism. The phage vehicle
incorporated dextran iron (Fe) with Fenton reaction capabilities.
H[2]O[2] is generated through the Lox catalytic reaction, amplifying
the H[2]O[2] supply for dextran iron‐based chemodynamic therapy (CDT).
Drawing inspiration from the erythropoietin (EPO) biosynthetic process,
an EPO enhancer is constructed to impart the EPO‐Keap1 plasmid (DNA)
with tumor hypoxia‐activated functionality, disrupting the redox
homeostasis of the TME. Lox consumes local oxygen, and positive
feedback between the Lox and the plasmid promotes the expression of
kelch ECH Associated Protein 1 (Keap1). Consequently, the
downregulation of the antioxidant transcription factor Nrf2, in synergy
with CDT, amplifies the oxidative killing effect, leading to tumor
suppression of up to 78%. This study seamlessly integrates adaptable T4
phage vehicles with bio‐intelligent plasmids, presenting a promising
approach for tumor therapy.
Keywords: antioxidant braking, chemodynamic therapy, fenton reaction,
Nrf2 signal pathway, phage display technology
__________________________________________________________________
A Fenton reaction function phage vehicle TLDF that flexibly modulates
enzyme numbers to remodel the TME. Lox molecules deplete lactic acid,
reorganizing the tumor's metabolism and producing H[2]O[2] to boost
dextran iron‐based CDT. Lox consumes oxygen and positive feedback
between the enzyme and EPO‐Keap1 plasmid, which synergizes with CDT to
enhance the oxidative killing effect.
graphic file with name ADVS-11-2308349-g003.jpg
1. Introduction
In 2018, the Nobel Prize in Chemistry recognized phage display
technology for its pioneering contributions to protein engineering and
drug advancement.^[ [42]^1 ^] Phage display technology stands as a
powerful tool that facilitates the display of exogenous proteins on the
phage surface.^[ [43]^2 ^] Notably, T4 phage exhibits a substantial
protein binding capacity with 1025 display sites, rendering them
exceptional vehicles for protein transport.^[ [44]^2a ^] In addition,
exogenous proteins can assemble on T4 capsids with remarkable
structural and functional integrity.^[ [45]^3 ^] The architecture of T4
is decorated with two non‐essential proteins: a small outer capsid
protein (Soc, 870 copies/head) and a highly antigenic capsid protein
(Hoc, 155 copies/head).^[ [46]^4 ^] The N and C termini of Soc are
prominently exposed, enabling Soc to function as an adapter for
displaying foreign proteins.^[ [47]^5 ^] The freely assembled Soc on
the phage head demonstrates nanomolar affinity, streamlining the
protein loading process and saving time.^[ [48]^5 ^] Crucially, the
concentration of fusion protein assembly modulates the number of
proteins on the phage surface.^[ [49]^2a ^] This tunable feature
empowers researchers to adjust protein abundance to meet diverse
experimental requirements and applications.
Enzymes catalyzed specific chemical reactions precisely within
organisms, effectively transforming endogenous substrates into toxic
substances,^[ [50]^6 ^] and were widely applied in chemodynamic therapy
(CDT).^[ [51]^7 ^] As an emerging therapeutic approach, CDT has
achieved precise anti‐tumor effects.^[ [52]^8 ^] Nevertheless, the
depletion of endogenous H[2]O[2] limited the effectiveness of CDT.^[
[53]^9 ^] In contrast to normal tissues, lactic acid, as a crucial
energy source, is abundant in the tumor microenvironment (TME) with
concentrations (5‐20 µmol/g) surpassing those of glucose (1‐2
µmol/g).^[ [54]^10 ^] Mounting evidence suggested that tumor cells
derived energy from lactic acid, which played a pivotal role in tumor
growth, metastasis, and recurrence.^[ [55]^11 ^]
A strategy for depleting lactic acid to remodel the TME and achieve
continuous catalytic H[2]O[2] production will significantly enhance the
effectiveness of CDT.^[ [56]^12 ^] Additionally, the highly reductive
TME also hindered the therapeutic effect of CDT.^[ [57]^13 ^] The
active metabolic activity of tumor cells has many sources of reactive
oxygen species (ROS),^[ [58]^14 ^] however, nuclear factor E2‐related
factor 2 (Nrf2) upregulates metabolic programs to detoxify ROS as a
compensatory mechanism.^[ [59]^15 ^] In healthy tissues, kelch ECH
Associated Protein 1 (Keap1) regulates Nrf2 activity,^[ [60]^16 ^]
whereas the gain of Nrf2 or loss of the Keap1 function is prevalent in
tumor tissues.^[ [61]^17 ^] The Nrf2 signaling pathway scavenges
ROS‐induced oxidative stress, thereby limiting the applicability of
CDT.^[ [62]^15 ^] TME‐responsive nanomaterials find extensive
application in tumor therapy.^[ [63]^17b ^] For example,
hypoxia‐sensitive drugs, such as Tirapazamine, have been developed to
precisely target tumor cells.^[ [64]^18 ^] Consequently, the
hypoxia‐activated smart regulatory plasmid disrupting tumor redox
homeostasis is highly prospective for augmenting therapeutic
efficacy.^[ [65]^13 ^]
Drawing inspiration from the advantages of phage display technology, we
introduced an in vitro phage display technology to construct a
T4‐Lox‐DNA‐Fe (TLDF) protein vehicle for enhanced CDT (Scheme [66]1 ).
Regarding the elevated demand for lactic acid in tumors, we
strategically presented a customized Soc–Lox fusion enzyme, with Soc
serving as an aptamer, displayed on the T4 phage surface (T4‐Lox, TL).
The molar ratio of the fusion protein and the binding site can be
adjusted for flexible regulation of protein abundance. Compared to Hoc,
both the N‐ and C‐termini of Soc are well exposed, allowing for
flexible adjustment of enzyme position and conformation. Dextran iron,
a drug for iron‐deficiency anemia, camouflaged and imparted phage
vehicles with a Fenton catalytic function. Utilizing endogenous lactic
acid as a “key”, the Lox enzymatic process was activated, remodeling
the tumor's energy metabolism and generating H[2]O[2] to enhance
dextran iron‐based CDT. Drawing inspiration from the erythropoietin
(EPO) biosynthetic process, we incorporated a double‐tandem EPO
enhancer into the EPO‐Keap1 plasmid, conferring it with hypoxia
activation capacity. This will enhance the controllability of gene
therapy and downregulated Nrf2. In vitro and in vivo results
demonstrated that the two‐pronged strategy of H[2]O[2]‐supplying CDT
and gene‐regulated antioxidant brakes disrupted redox homeostasis,
resulting in mitochondrial dysfunction in tumor cells. This study
introduced a tunable enzyme vehicle through phage display technology in
conjunction with bio‐intelligent plasmids to enhance CDT, thus
unveiling a novel anti‐tumor strategy.
Scheme 1.
Scheme 1
[67]Open in a new tab
Schematic presentation of the dual‐key activated TLDF probe to disrupt
tumor redox homeostasis for enhanced CDT.
2. Results and Discussion
2.1. Preparation and Characterization of TLDF
For the construction of an enzyme delivery system, T4 phage with the
Soc capsid gene deletion was employed as a nanocarrier (T4^△Soc denoted
as T4). The deletion of the Soc capsid protein did not hinder the
recognition and infiltration of T4 into the Escherichia coli (E. coli)
BL21 strain (Figure [68]S1, Supporting Information). The amplified T4
underwent purification through sucrose density gradient centrifugation
to eliminate impurities, including immature phages and bacterial debris
(Figure [69]1a). The results of transmission electron microscopy (TEM)
revealed that unassembled tail tubes of T4 were predominantly
concentrated in sucrose with densities below 15%, while mature T4 was
observed between 15% and 50% (Figure [70]1a). The matured T4 phage was
≈229 nm long and 106 nm wide (Figure [71]1b) and the plaque‐forming
unit (pfu) was determined by the double‐layer agarose method (Figure
[72]S2, Supporting Information). Lox enzymes positioned at the C
terminus of Soc were displayed on the surface of T4 via affinity
binding of Soc to phage capsid (T4‐Lox, TL). In comparison to the T4
phage, TL demonstrated a slight increase in dimensions, measuring a
length of 240 nm and a width of 112 nm (Figure [73]S3, Supporting
Information). Both T4 and TL exhibited surfaces with significant
negative charges, as shown in Figure [74]1c. To achieve plasmid
loading, TL was modified with the cationic polymer poly‐L‐Lysine. The
zeta potential of poly‐L‐Lysine‐functionalized TL (TLL) transitioned
from negative (−27.8 mV) to positive (32.0 mV) and the charge stability
of prepared TLL was related to the incubation time (Figure [75]S4,
Supporting Information). Following plasmid adsorption, the zeta
potential reverted to −27.4 mV (Figure [76]1c), signifying the
successful preparation of T4‐Lox‐DNA (TLD). Ultimately, to prevent
plasmid degradation, minimize the immunogenicity, and provide iron for
the Fenton reaction, TLD was functionalized with dextran iron oral
fluid (a drug for iron deficiency anemia) to form a T4‐Lox‐DNA‐Fe probe
(TLDF). The results revealed that the assembly process of dextran iron
was time‐dependent (Figure [77]S5, Supporting Information). A layer of
dextran iron with a thickness of 20 nm was formed on the TLD after
assembly for 2 h (Figure [78]1d,e). In comparison to other
iron‐containing nanoparticles, the preparation process of TLDF required
only a brief stirring, which would facilitate the retention of Lox
proteins. In addition, the hydrodynamic diameters of probes were
consistent with the results of TEM (Figure [79]1f). The in vitro
stability experiment of TLDF was performed. It exhibited that the
hydrodynamic diameters at 4 °C remained stable over a week (Figure
[80]S6, Supporting Information).
Figure 1.
Figure 1
[81]Open in a new tab
Preparation and characterization of TLDF vehicle. a) Purification of T4
phage by sucrose density gradient centrifugation and corresponding
phage TEM images (Scale bar: 100 nm). b) TEM images of T4 and d,e)
TLDF. c) Zeta potentials of T4, TL, TLL, TLD, and TLDF and f)
corresponding hydrodynamic diameter (n = 4). g) HAADF‐STEM image of
TLDF and elemental mapping images. h) Energy dispersive spectra TLDF.
i), j) XPS spectrum of TLDF and high‐resolution scanning energy spectra
of iron.
UV–vis spectra demonstrated that the characteristic absorption peaks
remained unchanged during the probe preparation (Figure [82]S7,
Supporting Information). The high‐angle annular dark field
(HAADF)‐mapping images and energy‐dispersive X‐ray spectroscopy (EDS)
disclosed that the TLDF primarily consisted of N, Fe, P, O, and C
(Figure [83]1g,h). Among them, the P element could be attributed to the
exogenous plasmid, and the Fe element was mainly distributed around the
phage. Simultaneously, the chemical composition and chemical states of
TLDF were measured by X‐ray photoelectron spectroscopy (XPS). As shown
in Figure [84]1i, the TLDF was mainly composed of C, N, O, P, and Fe
elements. The high‐resolution spectra of iron showed that the binding
energies of Fe^2+ 2p[3/2] and Fe^2+ 2p[1/2] were 709.14 and 722.18 eV,
respectively, while the binding energies of Fe^3+ 2p[3/2] and Fe^3+
2p[1/2] were located at 710.88 and 724.28 eV, respectively
(Figure [85]1j). The valence state of iron ions in the TLDF consisted
of divalent and trivalent in ratios of 26.03% and 73.97%, respectively
(Table [86]S1, Supporting Information). The loading content of iron on
the TLDF was quantitatively determined using atomic absorption
spectrophotometry and found to be 803 µg L^−1 (5 × 10^11 pfu, Figure
[87]S8, Supporting Information).
2.2. Construction and Expression of Soc–Lox Fusion Protein
Lactic acid assumes a pivotal role in the processes of tumor
proliferation, metastasis, and recurrence. In response to the
heightened demand for lactic acid in tumor tissues, we advocate for the
construction of a lactate oxidase (Lox) delivery vehicle to disrupt the
energy source of tumor cells. Both the N and C terminus of Soc are well
exposed, allowing Soc suitable as an adapter to display foreign
proteins on the T4 capsid. The Lox nucleotide sequence (GenBank:
[88]D50611.1) derived from Aerococcus viridans was inserted into the
upstream or downstream sequence of the Soc gene to construct the
Lox–Soc or Soc–Lox open reading frame (ORF).^[ [89]^19 ^] Surprisingly,
the fusion protein exhibited a lack of catalytic activity when the Lox
coding sequence was positioned at the N‐terminal of Soc (Lox–Soc,
Figure [90]2a). In contrast, Soc–Lox displayed high catalytic activity.
The tFold program was employed to construct a 3D structure model of the
fusion protein sequence from scratch (Figure [91]2b). Insights from 3D
modeling revealed that a noticeable difference between the Lox–Soc and
Soc–Lox fusion proteins lay in their exhibited reverse symmetry, and
the Lox–Soc fusion protein had 1–2 smaller alpha helices in the Soc
protein region, whose steric hindrance would affect the flexibility.
The Soc–Lox nucleotide sequence was depicted in Figure [92]S9a
(Supporting Information) and validated through DNA sequencing using a
T7 terminator primer (Table [93]S2, and Figure [94]S9b, Supporting
Information). After that, Soc–Lox was ligated into the pET‐32a vector
and expressed in the E. coli BL21 (DE3) strain.
Figure 2.
Figure 2
[95]Open in a new tab
Construction and expression of Soc–Lox fusion protein. a) Schematic
diagram of Soc–Lox plasmid construction and protein expression. b) The
Lox–Soc (green color) and Soc–Lox (yellow color) fusion protein 3D
structure model. c) Grayscale values of Soc–Lox protein expression
induction by different IPTG concentrations. d) Soc–Lox protein
purification. e) Michaelies–Menten and f) Lineweaver–Burk plot fitting
of H[2]O[2] production by the Soc–Lox fusion protein (n = 3). g)
SDS‐PAGE analysis Soc–Lox protein display onto phage and h) grayscale
values (n = 3). i) WB examination of the Soc–Lox fusion protein
displayed on phage. j) Quantification of the number of Soc–Lox fusion
proteins on each phage (n = 3). k), l) Schematic of TMB oxidation by
H[2]O[2] production by different amounts of TL and pictures of oxTMB
solution (45 min). m) UV–vis absorption spectra of oxTMB and n)
oxidation 2,6‐dichloroindophenol. o) DCFH‐DA fluorescence signals in
the cells after treatment with different concentrations of TL.
Isopropyl β‐D‐thiogalactoside (IPTG), functioning as a lactose analog,
was employed to regulate the expression of Soc–Lox protein. Various
concentrations of IPTG (0–1 mm) and induction time (3, 6, 12, and 24 h)
were performed (Figure [96]S10a, Supporting Information). A positive
correlation was observed between protein expression and IPTG
concentration during the brief induction period (Figure [97]S10b,c,
Supporting Information). Nevertheless, beyond 12 h, protein expression
exhibited a nearly independent relationship with IPTG concentration
(Figure [98]S10d, Supporting Information), and lower concentration
(0.1 mm) was determined to be effective in attaining elevated levels of
protein expression (Figure [99]2c; Figure [100]S10e, Supporting
Information). The Soc–Lox fusion protein with a His‐tag was purified
using the Ni‐NTA resin affinity chromatography method.^[ [101]^20 ^]
Miscellaneous proteins were progressively eliminated by adjusting the
imidazole concentration, where a purer fusion protein band was observed
at 70 kDa when the imidazole concentration reached 200 mm
(Figure [102]2d). Employing a His‐tag antibody for western blot (WB)
analysis unveiled a distinct signal indicative of antibody binding on
the PVDF membrane (Figure [103]S11, Supporting Information).
Additionally, mass spectrometry of the purified protein identified a
signal peak at 66.4 kDa (Figure [104]S12, Supporting Information).
These results confirmed that the Soc–Lox protein has been successfully
constructed, expressed, and purified.
In assessing the catalytic efficacy of the Soc–Lox fusion protein, we
determined the steady‐state kinetic parameters for Soc–Lox. Various
concentrations of lactic acid (1.0, 2.5, 5.0, 10, 20, 40, and 50 mm)
were employed as substrates. We measured the production rate of
H[2]O[2]during the reaction, and the substrate concentration, along
with the corresponding reaction rate, was fitted using the
Michaelis–Menten equation (Equation [105]1, Figure [106]2e).
Additionally, Lineweaver‐Burk fitting (Equation [107]2) was carried out
by plotting the reciprocal of the substrate concentration against the
reciprocal of the reaction rate (Figure [108]2f). The results indicated
that the Soc–Lox fusion protein exhibited a Michaelis constant (K [m])
of 5.12 mm and a maximum reaction velocity (v [max]) of
16.45 µM min^−1.
[MATH: v=vmax∗s/km+s
:MATH]
(1)
2.2. (2)
Soc, functioning as an aptamer for in vitro enzyme display, presents
more substantial advantages when compared to traditional loading
technologies. This includes a greater loading capacity and increased
flexibility in modulation potentials. To identify the optimal assembly
molar ratio of Soc–Lox for the enzyme display, we calculated the
molarity of the Soc site according to Avogadro's constant
(Equation [109]3). Soc–Lox fusion proteins, featuring diverse molar
ratios (ranging from 1:0 to 1:50), were introduced to the enzyme
display, and a gradual rise in the display of Soc–Lox protein on the
phage as the molar ratio increased (Figure [110]2g). Notably, the
maximum number of displayed proteins was attained at a molar ratio of
1:40, as depicted in Figure [111]2h. The displayed Soc–Lox was
specifically detected by WB, revealing that the antibody signal
progressively increased with an elevated molar ratio, reaching a
substantial binding signal at a molar ratio of 1:40 (Figure [112]2i).
These experimental findings substantiated the successful display of
Soc–Lox. T4 as a vehicle can accurately quantify the number of enzyme
molecules displayed on each phage based on the number of gp23* (930
copies) (Equation [113]4). The quantification of Soc–Lox indicated that
720 Lox molecules were displayed at a 1:40 molar ratio
(Figure [114]2j), which was below the maximum assembly capacity of 870.
It was also observed that the quantity of Soc–Lox fusion proteins on
phage remained relatively constant even with an increased molar ratio
of 1:50 (729). This phenomenon could be attributed to spatial site
resistance, which restricted the number of Soc–Lox bindings. In
conclusion, the developed phage delivery vehicle enables precise
control of the protein number by adjusting the feeding ratio within a
specified range. The Soc aptamer exhibited high efficiency, loading
enzymes within a mere 30 min without the need for additional reagent
assistance.
[MATH: Mole(Soc)=N∗870/NA :MATH]
(3)
where N was the number of T4, 870 was the number of Soc sites, and N[A]
was Avogadro's constant.
[MATH: Number(Soc−Lox)=Grayscale(Soc−Lox)/Grayscal<
mi mathvariant="normal">e(gp23∗)∗
930 :MATH]
(4)
where Grayscale[(Soc–Lox)] and Grayscale[(gp23*)] were the grayscale of
Soc–Lox and the major capsid protein gp23* corresponding to the
SDS‐PAGE result, respectively, and 930 was the number of gp23*.
The Soc–Lox displayed on the phage was designed to deplete lactic acid
and produce H[2]O[2]. The catalytic properties were assessed
(Figure [115]2k; Figure [116]S13a, Soc‐Lox), and the content of
H[2]O[2]exhibited a remarkable increase with the TL number
(Figure [117]2l). In the absence of Lox, however, no H[2]O[2] was
detected (Figure [118]S13b, Supporting Information). The increase in
the absorbance of oxTMB at 660 nm indicated that TL enzymatic activity
produced H[2]O[2] in a time‐dependent manner (Figure [119]2m). To
monitor the redox interaction of TL with lactic acid, phenoxazine
methyl sulfate (PMS, a yellow solution) was employed as an electron
transfer agent. This agent is capable of receiving transferred
electrons within the enzymatic reaction and transferring them to
2,6‐dichloroindophenol (DCIP, a blue solution). The color of DCIP
gradually faded and completely disappeared after 5 min (Figure
[120]S14), and the absorbance of DCIP at 600 nm steadily decreased
(Figure [121]2n). 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA)
was employed as an indicator to track the catalytic activity of TL in
cells. As shown in Figures [122]2o and [123]S15 (Supporting
Information), the green fluorescence signal in 4T1 cells was increased
with the TL, indicating the enzyme vehicle retained its catalytic
potential in the complicated intracellular environment.
2.3. Construction of a Recombinant DNA Regulatory System with an
Oxygen‐Sensing Function
While tumor cells exhibit active metabolic activity with numerous
sources of ROS, Nrf2 stands out as a key transcription factor
responsible for intracellular antioxidant activity.^[ [124]^21 ^] Nrf2
regulates the expression of antioxidant genes, thereby shielding tumors
from ROS attacks in tumor tissues. In normal physiological conditions,
Keap1 is responsible for tightly regulating Nrf2 activity, whereas the
prevalence of Nrf2 gain or Keap1 loss is observed in tumor tissues
(Figure [125]3a). Hypoxia will enhance EPO gene transcription levels
under physiological conditions.^[ [126]^22 ^] Inspired by this, a
recombinant plasmid with an oxygen‐sensing function was constructed.
This construction involved the insertion of a tandem repeat EPO
enhancer upstream of the SV40 promoter of the pGL4.73 plasmid
(Figure [127]3a). Under normal tissues, the EPO enhancer remained
silent and did not activate downstream SV40 promoter transcription.
Figure 3.
Figure 3
[128]Open in a new tab
Construction of a recombinant DNA regulatory system with an
oxygen‐sensing function. a) The working principle of oxygen‐sensing
EPO‐Keap1 plasmid. b) Agarose gel results of probe loading EPO‐Keap1
plasmids. c) RV primer 3 and RV primer 4 PCR amplification bands and d)
corresponding grayscale values (n = 3). e) The result of naked
EPO‐Keap1 plasmid and TLDF probe digestion by DNase I enzyme. WB
analysis of Nrf2 protein degradation by different plasmids at 24 h f)
or 48 h g) in an anoxic environment. Under normoxia h) or hypoxia i)
conditions degradation of intracellular Nrf2 by different plasmids.
However, the EPO enhancer will activate in the hypoxic TME, resulting
in the upregulation of downstream Keap1 gene expression (GenBank:
[129]AB020063.1) and downregulation of the Nrf2 signaling pathway
through the Keap1‐Nrf2 axis.
We investigated the optimal assembly conditions of EPO‐Keap1 onto the
surface of TL (5 × 10^11 pfu) with varying plasmid contents (2, 3, 5,
6, 8, and 10 µg). Agarose gel electrophoresis results indicated a
gradual increase in fluorescence signal with increasing plasmid
feeding, reaching a maximum at 6 µg (Figure [130]3b). The prepared TLD
probe served as the template and PCR amplification was conducted with
reporter vectors primer 3 (RV primer 3) and primer 4 (RV primer 4) of
pGL4.73 plasmid. The position of amplification bands was located
between 2 and 3 Kbp, which was consistent with the anticipated size of
2832 bp (Figure [131]3c), indicating that the EPO‐Keap1 plasmid had
been successfully loaded on the poly‐L‐lysine modified TL. Quantitative
analysis revealed the highest amount of amplified nucleic acid sequence
at a feeding concentration of 6 µg (Figure [132]3d). Additionally,
Keap1 protein‐specific primers were also designed (Table [133]S2,
Supporting Information), and agarose gel electrophoresis further
supported the aforementioned results (Figure [134]S16, Supporting
Information). During gene therapy, exposed DNA molecules were
susceptible to rapid degradation by nucleases in the serum, leading to
the loss of their function. To address this issue, the TLD was modified
by dextran iron (TLDF). In vitro nuclease degradation experiments using
DNase I demonstrated that the naked DNA was completely degraded, while
the plasmid in TLDF remained intact (Figure [135]3e).
To validate the inhibition of the Nrf2 signaling pathway by the
EPO‐Keap1 plasmid in cells, several plasmids were designed, including
pGL4.73, pGL4.73‐EPO, pGL4.73‐Keap1, and pGL4.73‐EPO‐Keap1. WB results
under hypoxic conditions for 24 h showed no significant change in the
glycosylation‐modified Nrf2 (110 kDa) with different plasmid regulation
(Figure [136]3f), indicating that the plasmid had no immediate effect
(Figure [137]S17, Supporting Information), while, with the treatment
time extended to 48 h, significant degradation of Nrf2 proteins was
observed in the EPO‐Keap1 group (Figure [138]3g). The oxygen‐sensing
capability of the plasmids was also investigated. Under normal oxygen
concentrations, the EPO enhancer remained inactive, and the EPO‐Keap1
plasmid exhibited no activity (Figure [139]3h). Conversely, in a
hypoxic environment, the EPO enhancer promoted Keap1 expression and led
to significant degradation of the Nrf2 protein (Figure [140]3i; Figure
[141]S18, Supporting Information).
2.4. TLDF Cascade Reaction Generation Hydroxyl Radicals
As a catalyst, dextran iron conferred phage vehicle with a Fenton
reaction ability. Hydroxyl radicals were generated by the TLDF in a
cascade‐like manner (Figure [142]4a). To determine the optimal
substrate concentration, TLDF was incubated with various concentrations
of lactic acid (0.5, 1.0, 2.0, 2.5, 5.0, and 10 mm). An increase in
lactic acid concentration led to a gradual rise in H[2]O[2] production,
reaching a plateau of 41 µm at 5 mm (Figure [143]4b). Metal ions are
known to bind to the enzyme's active site, potentially inhibiting
enzyme activity. There was a minimal disparity in H[2]O[2] production
at equivalent substrate concentrations, suggesting dextran iron has
negligible influence on Lox (Figure [144]4c). A distinct oxidized TMB
(oxTMB) absorption peak emerged through a cascade‐like reaction
(Figure [145]4d). Fe^3+ ions were reduced to Fe^2+ by GSH, and the
remaining GSH gradually declined, resulting in a decrease in UV–vis
absorption at 412 nm (Figure [146]4e). In addition, total glutathione
(GSH) levels in cells were also investigated. Intriguingly, the TL +
Lac group demonstrated the ability to deplete intracellular GSH even in
the absence of Fe elements (Figure [147]4f). Conceivably, a fraction of
the GSH could have been consumed by the H[2]O[2] produced by the TL,
and the TLF + Lac group exhibited a significant depletion of GSH, which
could be associated with the valence change of Fe^3+/Fe^2+
(Figure [148]4f). The ability of the TLDF to deplete GSH and enhance
•OH production was additionally investigated using methylene blue (MB).
The absorption peak at 650 nm in the TLDF + Lac group was markedly
diminished in the presence of GSH (Figure [149]4g). Subsequently,
5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) served as the capture reagent
to identify the type of ROS through electron spin resonance
spectroscopy (ESR). As shown in Figure [150]4h, a 1:2:2:1 DMPO‐OH
signal peak was seen in the TLF + Lac group, indicating that H[2]O[2]
produced by Lox could potentially generate •OH with dextran iron.
Figure 4.
Figure 4
[151]Open in a new tab
TLDF probe cascade reaction generation hydroxyl radicals. a) The
generation process of •OH in a cascade‐like manner. b) H[2]O[2]
production by the TLDF with different lactic acid concentrations
(n = 3). c) The amount of H[2]O[2] production by the TLD and TLDF with
lactic acid at 5 mm (n = 3). d) UV–vis absorption spectra of oxTMB. e)
The amount of GSH remaining in the solution after GSH incubation with
TLDF for different times and f) total GSH content in 4T1 cells after
incubation with different probes (n = 3). g) MB detection of •OH
production by different probes. h) Electron spin resonance spectra of
•OH trapped by the DMPO. i) Changes in dissolved oxygen concentration
in solution during Lox enzymatic reactions. WB detection j) Nrf2 and k)
HO‐1 protein content after different treatments. l) Fluorescence images
of DCFH‐DA‐stained 4T1 cells incubated with different probes.
The enzymatic reaction can effectively interact with the EPO‐Keap1
plasmid. Throughout the enzymatic reaction, there was an observed
decline in dissolved oxygen by 1.9 mg L^−1 (Figure [152]4i), fostering
a positive feedback regulation of EPO‐Keap1 expression. The degradation
of Nrf2 was found in the TLDF + Lac group compared with the TLDF group
(Figure [153]4j; Figure [154]S19, Supporting Information). To verify
this hypothesis, tris(4,7‐biphenyl‐1,10‐o‐phenanthroline) ruthenium
dichloride served as an oxygen‐sensitive dye to detect intracellular
oxygen concentration. The high concentration of oxygen quenched the
fluorescence in the TLDF group, whereas a prominent fluorescent signal
was observed in the TLDF + Lac group (Figure [155]S20, Supporting
Information). Nrf2 regulates the expression of heme oxygenase‐1 (HO‐1),
providing resistance to “ferroptosis” in tumor cells. The notable
down‐regulation of intracellular HO‐1 was attributed to a reduction in
Nrf2 in the TLDF + Lac group (Figure [156]4k; Figure [157]S21,
Supporting Information). The synergistic “two‐pronged” strategy
involving the Fenton reaction (an increase of free radicals) and
EPO‐Keap1 (inhibition of free radical scavenging) in TLDF was assessed
using DCFH‐DA. A more intense fluorescent signal was observed in the
TLDF + Lac group, indicating that the EPO‐Keap1 plasmid suppressed the
expression of antioxidant genes, leading to the accumulation of •OH in
the cells (Figure [158]4l).
2.5. TLDF Phagocytosis and Therapy In Vitro
Chlorin e6 (Ce6)‐labeled TLDF (TLDF@Ce6) was incubated with 4T1 cells
for different times (0, 2, 4, 6, and 12 h) to investigate the mechanism
of probe entry into cells. The probe gradually permeated the cell
membrane, reaching its peak fluorescence intensity after 6 h (Figure
[159]5a). The TLDF was observed in the 4T1 cells through the ultrathin
TEM section (Figure [160]5b), whereas no probe was observed in the
control group (Figure [161]5c). Atomic absorption spectroscopy was used
to detect the intracellular iron content and phagocytosis efficiency,
and the results showed that the iron ions peaked at 957.85 ng g^−1
(Figure [162]5d), with phagocytosis efficiencies of 4.60%, 7.39%,
9.00%, and 8.39% at 2, 4, 6, and 12 h, respectively (Figure [163]5e).
The optimal incubation period was determined to be 6 h. We further
investigated the mechanism of endocytosed probes using various cellular
pathway inhibitors, including chlorpromazine hydrochloride (CPZ,
clathrin inhibitor), wortmannin (PI3K‐AKT inhibitor), EIPA (Na^+‐H^+
exchange channel and micropinocytosis inhibitor), and treatment at 4 °C
(Figure [164]5f). As shown in Figure [165]5g, low temperature affected
the energy‐mediated phagocytosis process. Meanwhile, the blockage of
Na^+‐H^+ ion exchange channels and macropinocytosis (EIPA) greatly
reduced intracellular fluorescence. Therefore, TLDF nanocarriers
transported enzymes and plasmids into cells mainly through
micropinocytosis‐mediated endocytosis, and the transmembrane process
required energy participation.
Figure 5.
Figure 5
[166]Open in a new tab
TLDF phagocytosis and therapy in vitro. a) Fluorescence imaging of Ce6
after 4T1 cells incubation with TLDF@C probe at different times. b)
Ultrathin sections of 4T1 cells after incubation with or c) without the
TLDF. d) Atomic absorption spectroscopy detection intracellular Fe
content and e) percentage of phagocytosis after 4T1 cells incubation
with the TLDF at different times (n = 3). Fluorescence imaging f) and
intracellular mean fluorescence intensity g) of TLDF@C probes
phagocytosed by 4T1 cells treated with different cellular pathways
inhibitors (CPZ: chlorpromazine) (n = 3). h) Cell viability of HUVEC,
Huc‐MSC, and 3T3 cells after incubation with the TLDF (n = 5). i) Cell
viability of 4T1 after different treatments (n = 4). j) Fluorescence
imaging of Calcein‐AM/PI‐stained 4T1 cells after different treatments.
Fluorescence imaging of k) JC‐1 and l) C11‐BODIPY after 4T1 cells
treated by different probes. m,n) Flow cytometry detection apoptosis
cells after different treatments and percent of apoptosis cells
(n = 3).
In the course of transportation, the probes inevitably engage with the
circulatory system. Human umbilical vein endothelial cells (HUVEC),
human umbilical cord mesenchymal stem cells (HUC‐MSCs), and 3T3 cells
(mouse fibroblast) were utilized as model cells to investigate the
cytotoxicity of the probes. All cells demonstrated high viability at
elevated concentrations of TLDF (5 × 10^11 pfu mL^−1), indicating its
minimal harm to the vascular system or fibroblast (Figure [167]5h). The
anti‐tumor efficacy of TLDF on 4T1 cells was assessed. Lactic acid
played a pivotal role in initiating TLDF, unlocking the “Pandora's Box”
of H[2]O[2] generated by Lox, subsequently converted to •OH through a
cascade‐like process. However, Nrf2 upregulated antioxidant gene
expression (NQO1, GCLC, and SLC7A11), which counteracted the •OH
detrimental effects (Figure [168]5i). The antioxidant braking strategy
augmented the cytotoxic effect, resulting in a robust PI signal in the
nucleus (Figure [169]5j). The status of mitochondrial membrane
potential (∆ψ[m]) was detected by the JC‐1. The TL, TLF, and TLDF
groups induced depolarization of the mitochondrial membrane potential
in the presence of lactic acid (green fluorescence signal). The
downregulation of Nrf2 in the TLDF group significantly heightened
membrane depolarization, resulting in mitochondrial dysfunction
(Figure [170]5k).
Ferroptosis, a form of iron‐dependent cell death driven by lipid
peroxidation, was monitored by the C11‐BODIPY. As shown in
Figure [171]5l, the Lox enzymatic reaction did not elicit an indicative
of ferroptosis. However, dextran iron made the phage vehicle with
efficient Fenton catalytic ability, and the tumor cell membrane was
subjected to lipid peroxidation (TLF + Lac). The enzymatic reaction,
effectively interacting with the EPO‐Keap1 plasmid, disrupted redox
homeostasis, and C11‐BODIPY‐labeled fluorescence was more pronounced
upon stimulation with TLDF + Lac (Figure [172]5l). To demonstrate
ferroptosis‐induced tumor cell death of the TLDF, different ferroptosis
inhibitors, including deferoxamine mesylate (DFO, an iron chelating
agent), GSH, and N‐acetyl‐L‐cysteine (NAC, a precursor of GSH
biosynthesis) were added to investigate the effect on cell viability.
The results showed that the DFO significantly inhibited the therapeutic
effect of TLDF by chelating with iron ions released from the TLDF
(Figure [173]S22, Supporting Information). GSH overexpression in tumor
tissue maintains growth‐required redox homeostasis, and cell viability
was enhanced when GSH was supplemented compared with without inhibitor
group. NAC also exhibited the cytoprotective effect by indirectly
increasing GSH content. The fluorescence signals of Annexin‐V and PI
were analyzed using flow cytometry (Figure [174]S23, Supporting
Information). In a two‐pronged synergistic treatment strategy, the
percentage of necrotic cells increased from 7.23% to 8.65%
(Figure [175]5m). Importantly, the EPO‐Keap1 plasmid remained inactive
in the absence of lactic acid, resulting in no significant difference
in apoptosis between the TLF and TLDF groups (Figure [176]S24,
Supporting Information). The primary factor contributing to the
functionality of the designed EPO enhancer was its sensitivity to
oxygen concentration, enabling its operation only in a hypoxic
environment (Figure [177]5n).
2.6. Transcriptome Analysis of the Mechanism of TLDF‐Mediated Ferroptosis in
Cells
Transcriptomic analysis was conducted to achieve a comprehensive
comprehension of cellular pathways and potential molecular mechanisms.
Gene Ontology (GO) was employed to analyze the biological processes,
cellular components, and molecular functions of the differentially
expressed genes. Differential genes were found to be implicated in
diverse biological processes, such as cell apoptosis, mitochondrial
function, and DNA binding. The expression levels of 38 and 95 genes,
respectively, enriched in the cellular response to hypoxia and DNA
damage stimuli, were significantly altered (Figure [178]6a). KEGG
enrichment analysis revealed that 33 differentially expressed genes
were enriched as regulators of the ferroptosis pathway
(Figure [179]6b). Furthermore, 39 genes were identified to be involved
in the p53 signaling pathway, and 61 genes participated in the TNF
signaling pathway. This discovery supports previous research
highlighting the significance of the p53 and TNF signaling pathways in
ferroptosis.^[ [180]^23 ^] Examination of the top 20 signaling pathways
unveiled that around 12.86% of those genes were associated with the
regulation of ferroptosis.
Figure 6.
Figure 6
[181]Open in a new tab
Transcriptome analysis of 4T1 cells after treated by TLDF. a) Top 20
enrichment in GO term enrichment analysis and b) KEGG pathway
enrichment analysis of differentially expression genes after 4T1 cells
treatment with TLDF probe. c) Volcano plotting of transcript expression
in 4T1 cells induced by the TLDF. d) Schematic diagram of TLDF‐induced
changes in intracellular metabolism.
The RNA sequencing results showed that 9259 differentially expressed
genes were identified in 4T1 cells stimulated by the TLDF + Lac
compared with the control groups (P[adj] < 0.05 and |log[2] fold
change| > 1). Among them, 4136 genes exhibited up‐regulation, while
5123 genes underwent downregulation (Figure [182]6c). Tumor cells
predominantly rely on glycolysis for energy, and there is a significant
increase in the synthesis of the essential molecule lactic acid in
various solid tumors.^[ [183]^24 ^] Previously reported results have
shown that Lox can effectively deplete lactic acid and block the energy
source of tumor cells. In addition, lactate depletion strategies can
alter energy metabolism to enhance tumor therapeutic efficacy.
Transcriptome analysis demonstrated that glycolysis‐related enzymes,
such as hexokinase (HK1) and phosphofructokinase (PFKl,
p = 1.37 × 10^−8), responsible for catalyzing the production of
fructose 1,6‐diphosphate, were markedly downregulated (Figure [184]6d).
Concurrently, isocitrate dehydrogenase (IDH3α), an enzyme integral to
the tricarboxylic acid cycle, and succinyl‐CoA synthetase (SUCLA2),
involved in the production of citric acid, exhibited upregulation
(Figure [185]6d). According to these findings, it appeared that the
lactate depletion strategy has altered the metabolic profile of tumor
cells. This could be attributed to Lox exerting an inhibitory effect on
lactic acid energy metabolism, compelling tumor cells to derive energy
through the TCA cycle.
Transcriptome analysis unveiled both positive and negative regulation
of ferroptosis by the p53 signaling pathway. The upregulated expression
of genes, such as spermidine/spermine N1‐acetyltransferase 1 (SAT1),
arachidonic acid lipoxygenase 3 (ALOXE3), and SLC25A28, has been
associated with the promotion of lipid peroxidation (Figure [186]6d).
Conversely, p53 has been observed to suppress the expression of System
XC‐ (SLC7A11) by upregulating the downstream CDKN1A gene (p21,
p = 7.81 × 10^−7). In the Lox enzymatic reaction, the EPO enhancer
detected low oxygen levels and subsequently increased Keap1 expression.
The transcriptome analysis revealed a significant downregulation in the
expression of Nrf2‐regulated antioxidant genes, such as NAD(P)H:
quinone oxidoreductase 1 (NQO1), glutamate–cysteine ligase catalytic
subunit (GCLC), and SLC7A11 (Figure [187]6d). In conclusion, the
EPO‐Keap1 signaling pathway produced an “antioxidant braking effect”,
amplifying the oxidative environment, while TLDF could also modify the
metabolic pattern of tumor cells, inducing ferroptosis.
2.7. TLDF Safety Evaluation In Vivo
Diverse factors, including the immunogenicity and physicochemical
properties of probes, can lead to hemolysis. The hemolytic properties
of T4, TL, and TLDF were evaluated before in vivo testing. The
hemoglobin was spilled out from the cells in water, serving as a
positive control group (Figure [188]S25,Supporting Information inset).
In contrast, prepared T4, TL, and TLDF probes showed essentially
identical absorbance at 560 nm to the PBS group (Figure [189]S25a,
Supporting Information), indicating that they did not induce hemolysis.
The morphology of erythrocytes remained unaltered, maintaining their
distinctive two‐sided concave structure (Figure [190]S25b, Supporting
Information). This result indicated that TLDF probes were safe for
intravenous administration.
To evaluate both short‐term and cumulative toxicity of the TLDF,
healthy mice were randomly divided into three groups: PBS, a single
injection of TLDF, and multiple injections of TLDF (every five days,
for six injections in total). At the molecular level, alanine
aminotransferase (ALT), aspartate aminotransferase (AST), creatinine
(Crea), and urea nitrogen (Urea) were examined. Statistically, there
was no significant difference in the ALT and AST levels between the
experimental group and control group (Figure [191]S26a, Supporting
Information), both remained in the normal range. Additionally, Crea and
Urea Crea and Urea were effectively filtered by the kidneys’ glomeruli
and tubules (Figure [192]S26a, Supporting Information). These findings
revealed that the phage vehicle camouflaged with dextran iron was safe
for the kidneys and liver.
White blood cells (WBC) are the main component of the immune system,
crucial for the body's defense against infections and diseases. No
evidence of an inflammatory reaction was observed in the WBC
measurements following TLDF delivery, which remained within the normal
range (Table [193]S3, Supporting Information). There was a minimal
change in the number of erythrocytes and platelets, and no significant
release of hemoglobin was observed (Figure [194]S26b, Supporting
Information). In addition, other routine blood parameters, such as the
number of lymphocytes, monocytes, neutrophils, erythrocyte cumulative
pressure, and mean erythrocyte volume were within healthy ranges (Table
[195]S3, Supporting Information). The organ index was calculated for
critical organs such as the heart, liver, lung, and kidney. The results
indicated that the administration of TLDF did not result in any adverse
effects on organ function (Figure [196]S26c, Supporting Information).
Hematoxylin‐eosin staining (H&E), including the heart, liver, kidney,
spleen, lung, and small intestine was performed to assess the impact of
TLDF on tissue structure and cell morphology. These organs exhibited
normal morphology and well‐defined histology, without any signs of
organ damage, degeneration, or necrosis (Figure [197]S26d, Supporting
Information).
2.8. Immunogenicity and Tumor Targeting of TLDF In Vivo
The capacity of the TLDF nanocarrier to evade immune system recognition
was crucial for tumor targeting. Healthy mice were randomly divided
into two groups (n = 4) to explore the immunogenicity of TLDF. One
group received the TLDF injections (Figure [198]7a), while the other
group was injected with PBS. Spleen samples were collected after immune
stimulation, and the TLDF group showed no splenomegaly (Figure
[199]S27, Supporting Information). When exposed to exogenous antigens,
antigen‐presenting cells, particularly dendritic cells (DCs), will
upregulate the co‐stimulatory molecules CD80 and CD86. To explore the
expression of co‐stimulatory factors in antigen‐presenting cells, a
gating strategy was employed to eliminate interference from dead and
adherent cells (Figure [200]S28a, Supporting Information). The
percentage of CD11/CD80 and CD11/CD86 in the TLDF group decreased from
1.64% to 1.34% (Figure [201]7b; Figure [202]S28b, Supporting
Information) and 0.55% to 0.25% (Figure [203]7c; Figure [204]S28c,
Supporting Information), respectively, compared to the PBS group.
Helper T cells (CD3/CD4) and cytotoxic T cells (CD3/CD8) are also
essential components of the immune response. Spleen cells were
co‐stained with CD3, CD4, and CD8 antibodies to gain more insight into
the T lymphocytes (Figure [205]S29, Supporting Information). The
CD3/CD4 and CD3/CD8 cell populations increased by 0.83%
(Figure [206]7d) and 0.19% (Figure [207]7e), respectively, following
TLDF treatment. However, there was no significant difference in the
counts of antigen‐presenting cells (Figure [208]7f) and T lymphocytes
(Figure [209]7g). Furthermore, IgG1 is a crucial immunoglobulin that
plays a pivotal role in humoral immunity, contributing to pathogen
neutralization and complement system activation. The levels of
immunoglobulin IgG1 in the serum were detected by enzyme‐linked
immunosorbent assay (ELISA). The results showed that the content of
IgG1 did not significantly increase stimulated by the TLDF
(Figure [210]7h). Dextran iron was an effective disguise material,
making phage protein vehicles exhibit immune inertia.
Figure 7.
Figure 7
[211]Open in a new tab
Immunogenicity and tumor targeting of TLDF in vivo. a) Schematic
illustration showing the treatment steps and procedures for evaluating
the immunogenicity of TLDF probe in Balb/C mice. b,c) Representative
flow cytometry analysis of the population of DC maturation (CD11c/CD80,
CD11c/CD86) in the spleen after treatment with TLDF on day 30.
Representative flow cytometry analysis of the population maturation of
d) CD4^+ T cells (CD3/CD4) and e) CD8^+ T cells (CD3/CD8) in the spleen
after treatment with TLDF on day 30. f) Quantitative data of the
population of DC maturation (n = 4). g) Quantitative data of the
population of CD4^+ T cells and CD8^+ T cells (n = 4). h) ELISA
detection IgG1 content in serum after mice treatment with TLDF on day
30 (n = 4). i) Representative fluorescence images of 4T1 tumor‐bearing
mice at various time points post tail vein injection of TLDF@C. j)
Representative fluorescence images of major organs at different time
points and k) corresponding fluorescence intensities at various time
points post tail vein injection of TLDF@C (n = 3). l) Pharmacokinetics
of Fe during blood circulation in healthy mice after injection of the
TLDF (n = 3).
The Cy5.5‐labeled TLDF (TLDF@C) was used for imaging to investigate the
in vivo circulatory dynamics of the TLDF (Figure [212]7i, inset).
Following a 6 h intravenous injection, the fluorescent signal gradually
appeared at the tumor site and reached the peak fluorescent signal at
36 h (Figure [213]7i). To visualize the in vivo distribution of TLDF@C,
major organs, including the heart, liver, spleen, lung, kidney, small
intestine, and tumor were collected at different time points (0, 6, 24,
36, and 48 h). TLDF@C underwent a “first‐pass effect” attributed to the
liver's vital detoxification role, resulting in notable accumulation in
the liver (Figure [214]7j). The TLDF@C suffered a “first‐pass effect”,
leading to significant accumulation in the liver. However, after 48 h,
the signal in the liver gradually decreased (Figure [215]7k). The small
intestine also contributed to probe metabolism and aimed the excretion
of TLDF@C through excrement or urine (Figure [216]S30, Supporting
Information). The blood half‐life of TLDF was determined by the atomic
absorption spectrometer and calculated to be t [1/2] = 10.06 h
(Figure [217]7l). The hemodynamic parameters obtained from this study
provided valuable insight into the pharmacokinetic properties of the
TLDF probe.
2.9. Two‐Pronged Synergistic Treatment Strategy of TLDF In Vivo
A tumor therapy protocol was devised based on the metabolism of TLDF
(Figure [218]8a). Mice bearing 4T1 tumors were randomly divided into
seven groups, including PBS, T4, Lox, Fe, TL, TLF, and TLDF. The mice
were administered different probes through the tail vein every three
days. The changes in body weight and tumor volume of the mice were
monitored every other day, and the body weight exhibited a significant
decrease in the first two days. However, the body gradually exceeded
the pre‐treatment level (Figure [219]8b), probably due to the organism
requiring a recovery period following the administration of the
anesthetic drug. The lactic acid environment at the tumor site
triggered the TL to generate H[2]O[2], leading to a remarkable 52%
enhancement in tumor suppression (Figure [220]8c), and the TLF group
enhanced the tumor inhibition rate by 12% by converting H[2]O[2] into
highly toxic •OH. In a two‐pronged synergistic treatment strategy, the
TLDF group negatively regulated the Nrf2, resulting in effective tumor
growth inhibition with a tumor inhibition rate of up to 78%
(Figure [221]8c). The size of the dissected tumors (Figure [222]8d) and
weight (Figure [223]8e) in different groups were consistent with the
tumor volume growth curve, and the TLDF therapy regimen exhibited a
significant inhibitory effect on tumor growth (Figure [224]8f). H&E was
routinely employed for the pathological examination of tissues. The
results revealed nuclear enlargement and irregular and tightly packed
morphology in the control groups, indicating rapid proliferation of
tumor cells. In the TLDF group, however, the nuclei were wrinkled,
signifying substantial necrosis in the tumor tissues (Figure [225]8g).
Figure 8.
Figure 8
[226]Open in a new tab
In vivo synergistic therapy of TLDF. a) Experimental outline showing
the treatment procedures for evaluating the therapeutic outcomes in 4T1
tumor‐bearing mice. b) Body weight and c) tumor volume change after
administering different probes (n = 5). d) Photographs of tumor tissue
extracted from 4T1 tumor‐bearing mice after treatment with various
probes and e) corresponding tumor tissue weight (Scale bar: 1 cm;
n = 5). f) Photographs of 4T1 tumor‐bearing mice before (0 d) and after
(12 d) treatment with various probes. Representative g) H&E staining,
h) TUNEL staining, and i) Nrf2 immunofluorescence staining of tumor
sections collected from the mice receiving different treatments on day
3 (Scale bar: 25 µm). j) Quantitative data of the Nrf2 fluorescence
staining (n = 3).
TdT‐mediated dUTP Nick‐End Labeling (TUNEL) staining was additionally
conducted to assess the therapeutic impact on tumors. Negligible
fluorescence‐labeled signals in the PBS, T4, Fe, and Lox groups
indicated that these probes did not cause DNA damage to tumor cells
(Figure [227]8h). Compared with the TL and TLF groups, a strong TUNEL
signal was observed in the TLDF group, which was consistent with the
results of H&E staining. The expression levels of hypoxia‐inducible
factor (HIF‐1α) at the tumor site were examined by immunofluorescence
staining. The phage vector, camouflaged with dextran iron, effectively
evaded clearance, allowing the Lox enzymatic reaction at the tumor site
to induce an increase in HIF‐1α levels (Figure [228]S31, Supporting
Information). To explore the influence of the Nrf2 signaling pathway on
tumor therapy, we conducted immunofluorescence staining of tumor sites
using the Nrf2 antibody. Prominent expression of the Nrf2 protein in
whole tumor tissue, indicating a correlation with accelerated tumor
growth in the PBS group (Figure [229]8i). In the TLF group, ferroptosis
increased tumor cell lipid peroxidation and the intracellular Nrf2
antioxidant signaling pathway also activated, resulting in a
significant enhancement of fluorescence signal, which reduced the
therapeutic efficacy (Figure [230]8j). The TLDF exhibited a significant
reduction in Nrf2 protein expression through the EPO‐Keap1 plasmid.
This result further supported the notion that TLDF could compromise the
efficacy of antioxidant defense mechanisms in malignant cells and
heighten their vulnerability to therapeutic interventions.
3. Conclusion
In this study, we reported a novel TLDF bio‐intelligent nanoprobe using
T4 phage display technology and oxygen‐sensing plasmid to enhance CDT.
The TLDF nanoprobes exhibited “personalized tumor therapy”
characteristics upon delivery. First, a high concentration of lactic
acid environment as a “key” triggered the Lox enzymatic reaction,
leading to CDT through a cascade‐like reaction. Second, the hypoxia TME
acted as the other “key”, activating the bio‐intelligent EPO‐Keap1
plasmid to interfere with the Nrf2 signaling pathway. Moreover, during
the starvation therapy accompanied by reduced oxygen concentration,
positive feedback regulation of EPO‐Keap1 expression was achieved.
Transcriptomic analysis revealed that the TLDF synergistic treatment
reversed the energy metabolism of tumors. The two‐pronged strategy of
H[2]O[2]‐supplying CDT and gene‐regulated antioxidant brakes disrupted
redox homeostasis and enhanced the therapeutic effect. More
importantly, the TLDF exhibited good biocompatibility and low
immunogenicity at the molecular, cellular, and tissue organ levels. The
TLDF probe was the first to integrate phage enzyme display technology
with an adaptive regulatory plasmid. This strategy combined synthetic
biology and nanotechnology to provide a new idea for anti‐tumor
therapy.
4. Experimental Section
Materials
All chemicals and reagents were provided by commercial sources.
N‐hydroxysuccinimide (NHS),
1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDC),
chlorpromazine hydrochloride, wortmannin, and EIPA were obtained from
Shanghai Aladdin Biochemical Technology Co., Ltd. Chlorin e6 (Ce6) was
purchased from Shanghai Macklin Biochemical Co., Ltd. Mitochondrial
membrane potential assay kit (JC‐1) was obtained from Beyotime. Mouse
IgG1 ELISA kit was purchased from MULTISCIENCES (LIANKE) BIOTECH, Co.,
Ltd. DNA marker (100‐3000 bp), M9 minimal salts, 50× TAE buffer,
ElL‐ABTS chromogenic reagent kit, and peroxidase (Rz > 3.0) were
purchased from Sangon Biotech (Shanghai) Co., Ltd. Transcriptome
sequencing analysis (RNA‐seq) was performed by Benagen. PCR primer
synthesis and nucleic acid sequencing were synthesized by Beijing
Tsingke Biotech Co., Ltd.
Amplification of T4 Phage with Soc Capsid Protein Mutation
The Soc mutated T4 phage was kindly given by Prof. Song Gao from
Jiangsu Ocean University. 250 µL of overnight culture of E. coli BL21
and 2.5 × 10^6 pfu T4 phage was mixed with 150 mL of Luria–Bertani
medium (LB). The mixture was incubated at 37 °C and 100 rpm until the
solution became clear. The unlysed E. coli BL21 was removed through
centrifugation at 5 000 rpm for 30 min. The amplified phage was
collected by centrifugation at 34 000 g for 1 h in an ultrahigh‐speed
centrifuge (XE90, Beckman). To further remove impurities and cell
debris from the supernatant, 0.22 µm filter membranes were utilized.
The precipitate of T4 phage was subsequently re‐suspended in PBS,
followed by the preparation of 15% and 50% sucrose solutions for
further purification. The purified T4 was dialyzed overnight in Tris‐Mg
buffer (10 mm Tris‐HCl, 1 mm MgCl[2], and 100 mm NaCl).
Construction of Soc–Lox Recombination Plasmid
The Lox nucleic acid sequence (derived from Aerococcus viridans) was
optimized according to the codon preference of Escherichia coli, and
the optimized nucleic acid sequence was synthesized at General
Biosystems (Anhui, China) Co., Ltd. Soc. Lox was constructed as a
fusion protein. To improve the solubility of the fusion proteins, the
Soc–Lox and Lox–Soc nucleic acid sequences were amplified via forward
primer: 5’‐GACGACGACGACAAGGGTGGCTATGTTAATATTAAGACC‐3’ and reverse
primers: 5’‐GTCGACGGAGCTTTAATATTCATAACCATACGG‐3’ and ligated to pET‐32a
vector by seamless cloning kit (Vazyme, Nanjing). The recombinant
pET‐32a‐Soc‐Loc (Soc–Lox) and pET‐32a‐Lox–Soc (Lox–Soc) plasmids were
transformed into E. coli BL21 (DE3) for expression.
Soc–Lox Protein Display Process in Phage
T4 (5 × 10^10 pfu) was mixed with Soc–Lox protein at varying molar
ratios (1:0–1:50). After incubation at room temperature (RT) for
30 min, T4‐Lox (TL) was collected by centrifugation at 16 000 g for
40 min. The TL was re‐suspended in 50 µL PBS and detected by SDS‐PAGE.
Image J software was used to analyze the grayscale values of both the
Soc–Lox protein and the gp23* protein of the phage. The number of
Soc–Lox proteins displayed on each phage was calculated based on the
number of gp23* proteins.
Construction of EPO‐Keap1 Plasmid
The Keap1 nucleic acid sequence was derived from Mus musculus. To
improve expression in mammalian cells, the Keap1 nucleic acid sequence
was optimized by codon‐optimization and synthesized at General
Biosystems (Anhui, China) Co., Ltd. Keap1 nucleic acid sequence was
ligated to pGL4.73 by Hind III and Xba I digestion sites. The
oxygen‐sensing EPO enhancer was located at the N‐terminal of the Keap1
sequence. The constructed pGL4.73‐EPO‐Keap1 plasmid was transformed
into E. coli TOP10 stain for amplification.
Preparation and Characterization of TLDF
T4 (5 × 10^11 pfu) was incubated with Soc–Lox protein (molar ratio
1:40) at RT for 30 min. Prepared TL was collected through
centrifugation at 16 000 g for 40 min. A 2 mL of poly‐L‐lysine
(5 mg mL^−1) solution was used to re‐suspend the TL. After incubation
at RT for 2 h, the mixture was centrifuged at 16 000 g to obtain TLL.
The TLL was re‐suspended in deionized water and mixed with EPO‐Keap1
(6 µg) at RT for 30 min. The TLD was mixed with iron dextran (final
concentration of 0.5 mg mL^−1), and the mixture was agitated at 28 °C
for 2 h. Subsequently, the TLDF probes were washed twice with PBS and
stored at 4 °C.
Preparation of TLDF@Ce6 Probe
Ce6 (2 mg mL^−1) was dissolved in dimethyl sulfoxide (DMSO). EDC
(3.6 mg mL^−1) and NHS (2.6 mg mL^−1) were gradually added to the
reaction solution and stirred at 28 °C for 6 h. The activated Ce6‐NHS
was stored at −20 °C and without further purification. T4 (5 × 10^11
pfu) and Ce6‐NHS (100 µg mL^−1) were shaken for 6 h. The solution was
subjected to centrifugation at 16 000 g for 40 min. The obtained T4‐Ce6
was used for the preparation of TLDF@Ce6 probes following the
guidelines of TLDF.
Detection of Mitochondrial Membrane Potential
The overnight culture of 4T1 cells was washed twice with PBS and
incubated with the probes in a serum‐free medium for 6 h. The cells in
a complete medium containing lactic acid (2.5 mm) were incubated for
18 h. Cells were washed twice with PBS and added 1 mL of complete
culture medium and 1 mL of JC‐1 staining working solution, and then
incubated at 37 °C for 20 min. Upon completion of the incubation, the
cells were washed twice with JC‐1 staining buffer. Finally,
fluorescence microscopy was utilized to observe the intracellular JC‐1
signal.
The Blood Half‐Life of TLDF
To remove iron interference in red cells. Healthy Balb/c mice were
injected with PBS as a control group, and blood was taken at the same
time point as the experimental group. The weight of blood was recorded
and digested by HNO[3] (Guaranteed reagent, GR). The atomic absorption
spectrophotometer was used to measure the iron content in each sample.
The amount of iron in the blood at different time points in the
experimental group was calculated as follows.
[MATH: ContentofFe(μg/g)=FeE−FeCM(Blood) :MATH]
(5)
Fe[(E)] and Fe[(C)] represent the iron content of the experimental and
control groups at the same time. M[(Blood)] represents the weight of
blood.
The Synergistic Therapeutic Effect of TLDF In Vivo
Female Balb/c mice (five weeks) were subcutaneously injected with 4T1
cells (1 × 10^6) in the right hand side. Upon reaching a predetermined
tumor volume, the mice were randomly divided into seven groups: PBS,
T4, Lox, Fe, TL, TLF, and TLDF. A probe (6.25 × 10^12 pfu Kg^−1) was
administered via the tail vein every three days. Iron and Lox
concentration per administration was 803 µg L^−1 and 720
molecules/phage, respectively. After 72 h of treatment, one mouse from
each group was selected for euthanasia, and tumor tissues were immersed
in a 4% paraformaldehyde for H&E staining, TUNEL, and Nrf2
immunofluorescence staining. The mice's body weight and tumor volumes
were documented every other day. Upon completion of the treatment, all
mice were humanely euthanized, and the tumor tissues were extracted,
photographed, and weighed. All animal experiments were approved by the
Animal Experimental Ethics Committee of Huazhong University of Science
and Technology (IACUC Number: S904).
Statistical Analyses
All quantified data were expressed as mean ± standard deviation.
Statistical comparisons between the two groups were performed by a
two‐way Student t‐test and multiple comparisons by a one‐way ANOVA. ^*
p < 0.05, ^** p < 0.01, ^*** p < 0.001, and n.s. means no statistically
significant difference.
Conflict of Interest
The authors declare no conflict of interest.
Supporting information
Supporting Information
[231]ADVS-11-2308349-s001.pdf^ (2.6MB, pdf)
Acknowledgements