Journal of Controlled Release 350 (2022) 298–307

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Lipid nanoparticles produce chimeric antigen receptor T cells with

interleukin-6 knockdown in vivo
Jing-e Zhou 1, Lei Sun 1, Yujie Jia, Zhehao Wang, Tengshuo Luo, Jingwen Tan, Xiaoyan Fang,
Hongjia Zhu, Jing Wang, Lei Yu *, Zhiqiang Yan *
Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry
and Molecular Engineering, East China Normal University, Shanghai 200062, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Chimeric receptor T cells (CAR-T) can effectively cure leukemia; however, there are two limitations: a compli­
Chimeric antigen receptor T cells cated preparation process ex vivo and cytokine release syndrome (CRS). In this study, we constructed a lipid
CD3 antibody nanoparticle system modified by CD3 antibody on the surface, loading with the plasmid containing the com­
Lipid nanoparticles
bination gene of interleukin 6 short hairpin RNA (IL-6 shRNA) and CD19-CAR (AntiCD3-LNP/CAR19 + shIL6).
IL-6 shRNA
The system targeted T cells by the mediation of CD3 antibody and stably transfected T cells to transform them
Cytokine release syndrome
into CAR-T cells with IL-6 knockdown, thus killing CD19-highly expressed leukemia tumor cells and reducing
CRS caused by IL-6. In vivo experiments showed that AntiCD3-LNP/CAR19 + shIL6 could stably transfect T cells
and produce CAR-T within 90 days to kill the tumor. This significantly prolonged the survival time of leukemia
model mice and demonstrated the prepared LNP exhibited the same anti-tumor effect as the traditional CAR-T
cells prepared ex vivo. In this study, CAR-T cells were directly produced in vivo after intravenous injection of
the lipid nanoparticles, without the need of using the current complex process ex vivo. Additionally, IL-6
expression was silenced, which would be helpful to reduce the CRS and improve the safety of CAR-T therapy.
This method improves the convenience of using CAR-T technology and is helpful in further promoting the clinical
application of CAR-T.

1. Introduction
Chimeric antigen receptor T-cell (CAR-T) therapy has achieved
remarkable success in the treatment of hematological malignancies
[1–3], with several products currently approved by the FDA for acute B-
cell leukemia (B-ALL) [4,5], large B-cell lymphoma and multiple
myeloma [6,7]. However, the clinical application of CAR-T technology
is still limited by many factors [8], such as the complex preparation
process and cytokine release syndrome (CRS) [9].
Currently, obtaining CAR-T cells requires a complex process and
strict quality control [10]. A series of procedures, including blood
sample collection, separation, activation, transfection, amplification,
detection, preparation, transportation, and drug administration, need
up to about three weeks [10]. Additionally, the preparation of CAR-T
cells for clinical applications requires customization and following
GMP for each patient, making mass production difficult [11]. As CAR-T
therapy has high costs and technical barriers, it is difficult to be widely
used in clinical practice [12,13]. New technology is required for CAR-T
cell preparation to simplify its preparation process, avoid strict quality
control ex vivo, and reduce the technical barriers.
Another challenge for CAR-T therapy is cytokine release syndrome
(CRS) [9]. Studies have shown that about 60% to 80% of patients treated
with CAR-T may suffer from CRS [12]. In CRS, CAR-T cells are activated
after combining with the antigen of tumor cells, constantly expanding
and release copious amounts of cytokines, thus leading to a systemic
inflammatory response or even the death of the patient [14]. Tocilizu­
mab, a humanized IL-6 receptor monoclonal antibody, is often used
clinically to block IL-6 receptors and the associated signal transduction
[15–17], thus alleviating CRS [18]. However, Tocilizumab can often
cause neutropenia and severe bone marrow suppression, increasing the
risk of infection [19]. Thus, finding an effective way to control CRS for
CAR-T therapy is urgently required [20].
The Nano-delivery system can be used to deliver DNA to cells and
facilitate the direct production of CAR-T cells in vivo [21–23]. For

* Corresponding authors.
E-mail addresses: yulei@nbic.ecnu.edu.cn (L. Yu), zqyan@sat.ecnu.edu.cn (Z. Yan).
1
Jing-e Zhou and Lei Sun contributed equally to this work.

https://doi.org/10.1016/j.jconrel.2022.08.033
Received 6 April 2022; Received in revised form 8 August 2022; Accepted 16 August 2022
Available online 25 August 2022
0168-3659/© 2022 Elsevier B.V. All rights reserved.
J.-e. Zhou et al.
example, in 2017, Nat Nano [24] reported a type of nanoparticle that
encapsulated CAR DNA with cationic polymer and targeted T cells
through the mediation of CD3 antibodies and transformed them directly
into CAR-T cells. This study provided new insights into CAR-T treat­
ment. The investigator then developed nanoparticle-mediated delivery
of mRNA for CAR-T therapy that has entered the clinical phase
(NCT01837602 [25], NCT03060356 [26], NCT02277522 [27]). How­
ever, the delivery of CAR DNA with lipid nanoparticles (LNPs) remains
unreported. We choose LNP as gene delivery vector because it has more
advantages than cationic polymers. Cationic polymers have high mo­
lecular weight (most of them are more than 10KDa), which leads to their
poor biodegradability and easy accumulation in the body, thus causing
damage to tissues.
The LNP delivery systems had the following advantages: 1) LNP is
mainly composed of phospholipids, which has good drug-forming
property. It is easily metabolized and will not accumulate in the body
for a long time to cause toxicity. 2) The ionizable lipid used in LNP have
pH sensitivity. Its positive charge at low pH enables LNPs to escape from
endosomes, and its neutral charge at around pH 7.0 keeps LNPs stable in
the blood, avoids being quickly cleared in the blood. 3) LNP, as a gene
drug delivery system, has been marketed for several drugs, while
cationic polymeric system is still in the early stage of preclinical trials.
In this study, we prepared lipid nanoparticles (LNPs) modified by
CD3 antibody, which encapsulated plasmids containing human
interleukin-6 (IL-6) short hairpin RNA (shRNA) and the CAR gene. After
intravenous injection, the LNPs successfully transfected the T cells and
produced IL-6-knockdown CAR-T cells in vivo. The CAR-T cells showed
antitumor effects comparable to that of conventional CAR-T cells and
reduced the release of IL-6 and the incidence of CRS (Fig. 1). This
technology could avoid the complex preparation process ex vivo and
strict quality control of the traditional CAR-T technology, reduce the
technical barrier and production costs, and provide a more convenient
and practical protocol for CAR-T therapy.
2. Methods
2.1. Plasmid construction
All the plasmids used in this study were custom-cloned by Shanghai
Unicar-Therapy Biomed-Pharmaceutical Technology Co., Ltd.
Fig. 1. The design of LNPs and the production of CAR-T cells in vivo. After intravenous injection, the LNPs transfected the T cells and produced IL-6-knockdown
CAR-T cells in vivo.
299
Journal of Controlled Release 350 (2022) 298–307
The IL-6 shRNA sequence was inserted into the basic vector plasmid
PUT523 by ApaLI, PvuII, and XhoI endonuclease. The transposons were
also added to the plasmid genes, leading to a high transfection efficiency
and durable and stable gene expression. The primer was synthesized by
Shanghai General Bioengineering Co. Ltd. After the interference frag­
ment was ligated to the expression vector, the target plasmid was
transformed and identified by colony PCR, followed by DNA sequencing.
2.2. Cell lines
Raji-Luc cells, the K562 cell line, and human T cells were acquired
from Shanghai Unicar-Therapy Biomed-Pharmaceutical Technology
Co., Ltd. The K562 cells were transfected with lentivirus to obtain the
K562 cells expressing CD19. The Raji-Luc cell line and K562 cells were
cultured in complete Roswell Park Memorial Institute (RPMI) 1640
medium with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL
penicillin, and 100 μg/mL streptomycin.
2.3. MTAS-NLS peptide-DSPE-PEG synthesis
The sulfhydrylated microtubule-associated-nuclear localization
peptide Cys-MTAS-NLS (sequence.
CGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR)
was synthesized by Gil Biochemical Company. The addition of this
peptide into the LNPs could help the plasmid gene to enter the nucleus
and increase transfection efficiency [28]. Next, 10 mg of the nuclear
localization peptide Cys-MTAS-NLS was dissolved in 0.5 mL of 1X PBS
solution (pH 7.0). Then, 15.8 mg of Mal-PEG6-DSPE was dissolved in 1
mL of dimethylformamide (DMF), mixed with the peptide solution, and
stirred for 4 h. After the reaction was completed, the excess polypeptide
and DMF were removed by dialysis in ultra-pure water for two days
(Mw = 3.5 kDa). After freeze-drying, the target compound (DSPE-PEG-
MTAS-NLS) was obtained. The structure was characterized by IR spec­
trums and 1H NMR.
2.4. Synthesis and characterization of DSPE-PEG-CD3 antibody-targeted
lipid molecules
The anti-human CD3 antibody (Bioxcell.com; cat. no. BE0001–1)
was dissolved in 1 mL of NaHCO3 solution (pH = 8), and 4 mol. of DSPE-
J.-e. Zhou et al.
PEG2000-NHS was dissolved in dimethyl sulfoxide (DMSO). Then,
DSPE-PEG2000-NHS was slowly added to the CD3 antibody solution and
mixed for 4 h. After the reaction was completed, the solution was dia­
lyzed in ultra-pure water (cutoff molecular weight: 3.5 kDa) and freeze-
dried. The structures were characterized by HPLC and 1H NMR.
2.5. Preparation of AntiCD3-LNP/CAR19 + shIL6
The thin-film hydration and extrusion method [29] was used to
prepare the LNPs with IL-6 knockdown (LNP/CAR19 + shIL6). The
membrane materials, including DlinMC3, DOPE [30], Chol, PEG-DMG
[31], DOTAP, and DSPE-MTAS-NLS (molar ratio 1–20: 5–10: 1–10:
1–10: 1–5: 3), were weighed and dissolved in 2 mL of trichloromethane
as the lipid oil phase solution. Then, trichloromethane was gradually
removed using a rotary evaporator to obtain a film of uniform thickness.
Sodium acetate solution (pH = 4; 25 mM) was prepared as an aqueous
solvent, and the plasmid was dissolved in the solution, with a 1:10 M
ratio of the plasmid to the lipid materials. The prepared aqueous solu­
tion was added to the lipid film and mixed for 1 h. Then, the LNP/
CAR19 + shIL6NP/IL-6 was extruded through the nuclear pore mem­
branes of 400 and 200 nm using a micro-extruder. The LNP/CAR19 +
shIL6NP/IL-6 suspension was collected with a 10 kDa ultrafiltration
centrifuge tube and centrifuged at 6000 rpm for 30 min, and placed in
phosphate-buffered saline (PBS). Finally, DSPE-PEG2000-CD3 (in a
molar ratio of 1:10 to the plasmid) was added to the above solution, and
incubated for 4 h at 37 ◦ C to obtain the CD3 antibody-modified LNPs
with IL-6 knockdown (AntiCD3-LNP/CAR19 + shIL6), which was stored
at 4 ◦ C.
2.6. Nanoparticle characterization
The nanoparticle solution was diluted 50 times with PBS. The size,
polydispersity index (PDI), and zeta potential (ζ) of the LNPs were
determined by dynamic light scattering (DLS) (Malvern, UK). The
morphology of the LNPs was observed by transmission electron micro­
scopy (TEM). The nanoparticle samples were negatively stained with
phosphotungstic acid and observed after the samples were dried. The
prepared LNP solutions were evenly divided into two parts. DNase (5
μg/ug DNA) was added into one part to react at 37 ◦ C for 30 min, and
EDTA was used to stop the reaction. After DNase treatment, the un­
coated plasmid was decomposed, while the LNP-coated plasmid was
protected from degradation. The solution was centrifuged and the lower
layer was the LNP encapsulated plasmid. We determined the plasmid as
C (encapsulated). The other part was added with 5% TritonX-100, and
reacted at room temperature for 15 min, so that LNP was completely
broken to release the encapsulated plasmid. At this time, the total
plasmid content was determined as C (total). The encapsulation effi­
ciency was calculated by the following formula: Encapsulation effi­
ciency = encapsulated DNA/total DNA. We used BCA kit (Beyotime
Biotechnology) to detect the antibody loading of LNP. Briefly, free CD3
antibodies that do not bind to LNPs are removed by a sephadex column.
Then, AntiCD3-LNPs solution was mixed with methanol, and add 20 μL
into the 96 well plate. 200 μL of BCA working solution was added to each
well and stand at 37 ◦ C for 30 min. The absorbance of each well at 562
nm was measured by microplate reader, and the CD3 antibody con­
centration was calculated according to the standard curve. The antibody
loading rate is calculated as follows: Column-passing antibody/total
antibody*100%.
2.7. In vitro T-cell transfection using AntiCD3-LNP/CAR19 + shIL6
The AntiCD3-LNP/CAR19 + shIL6 nanoparticles and LNP/CAR19 +
shIL6 nanoparticles loaded with DiO fluorescent dye were prepared, and
the uptake of the two types of nanoparticles by T cells was observed with
a laser confocal microscope. The AntiCD3-LNP/CAR19 + shIL6 and
LNP/CAR19 + shIL6 loaded with GFP were incubated with human T
300
Journal of Controlled Release 350 (2022) 298–307
lymphocytes for 48 h, and the number of cells expressing GFP was
determined by flow cytometry to evaluate the transduction efficiency in
vitro.
2.8. Functions of AntiCD3-LNP/CAR19 + shIL6 in vitro
2.8.1. Cytotoxicity assay
We selected LNPs transduced T cells as the effector cells and K562 or
Raji cells as the target cells to evaluate the ability of killing tumor cells
The T cells were transfected with AntiCD3-LNP/CAR19 + shIL6, LNP/
CAR19 + shIL6, and AntiCD3-LNP/CAR19 (CD3 antibody modified
nanoparticles without knocking down IL-6) were incubated for 48 h with
CD19 overexpressed K562 target cells and Raji target cells in a ratio of
10:1, 8:1, 5:1, and 1:1, respectively. The in vitro cytotoxicity of CAR-T
cells was verified by the lactate dehydrogenase (LDH) method.
2.8.2. Cytokine secretion assay
To test the expression level of IL-6 mRNA, the T cells were trans­
fected with AntiCD3-LNP/CAR19, LNP/CAR19 + shIL6, and AntiCD3-
LNP/CAR19 + shIL6, followed by incubation with the target cells for 48
h; the co-incubated cells were collected to extract total RNA and detect
IL-6 expression by qPCR.
2.9. QPCR
To test the expression level of IL-6 mRNA, T cells were transfected
with AntiCD3-LNP/CAR19, LNP/CAR19 + shIL6, and AntiCD3-LNP/
CAR19 + shIL6, followed by incubation with the target cells for 48 h.
Then the co-incubated cells were collected, centrifuged, and the super­
natant was removed. Total RNA was extracted using TRIZOL Reagent
(Cat#15596–018, Life technologies, Carlsbad, CA, US) following the
manufacturer’s instructions. Then A 5 × Primescipt RT Master mix was
used to reverse-transcribe the extracted RNA to obtain cDNA. Total IL-6
expression was detected by qPCR. The primers were provided by
Shanghai Uni-car Biological Co. Ltd. The primer sequence for IL-6
amplification is:
Gene Name Sequences (5′ -3′ )
Forward: TAACCACCCCTGACCCAACCA
IL-6
Reverse: GCGCAGAATGAGATGAGTTGTCA
Forward: GGACAGGACCATATTGAGGGACA
GAPDH
Reverse: AGGAGTGAGTGGAAGACAGAATGGA
2.10. Mouse models
NOD-SCID IL-2 receptor gamma null (NSG) mice were injected with
5 × 106 Raji-Luc cells to construct acute lymphoblastic leukemia models.
After luciferase-induced fluorescence was observed (in approximately
one week), tumor-bearing mice were randomly divided into 8 groups (n
= 10). The drug was administered by tail vein injection at a dose of 5 μg/
g (calculated as the mass of the plasmid). The efficacy of LNPs was
compared with conventional CAR-T cell therapy by treating a group of
mice with a single dose of 5 × 106 △IL-6 CAR-T cells.
2.11. In vivo distribution of nanoparticles
The tumor-bearing mice were randomly divided into three groups,
namely, AntiCD3-LNP/CAR19 + shIL6 as the experimental group, LNP/
CAR 19 + shIL6 as the control group, and the blank normal saline group.
The in vivo tissue distribution of AntiCD3-LNP/CAR19 + shIL6 was
observed using the Small Animal In Vivo Imaging System (Perkin Elmer,
MA, USA) after tail vein injection of luciferase-labeled nanoparticles in
mice.
J.-e. Zhou et al. Journal of Controlled Release 350 (2022) 298–307

2.12. Quantification of CAR-T produced by AntiCD3-LNP/CAR19 +
shIL6 in peripheral blood
After injecting the nanoparticles into tumor-bearing mice, 0.1 mL of
peripheral blood was taken from the mice on the days 7th, 14th, 21st,
35th, and 90th, respectively. Then, white blood cells were separated
using a monocyte separation solution and erythrocyte lysis solution. The
isolated leukocytes labeled with anti-human CD3 (PE-Cy7), anti-human
CD4 (PE), anti-human CD8 (APC), and anti-human CD19 (FITC) were
detected by flow cytometry to determine the number of CAR-T cells.
2.13. Statistical analysis
All data are presented as the mean ± SD. Two groups of data were
analyzed by performing Student’s t-tests (two-tailed). More than three
groups of data were analyzed by performing analysis of variance
(ANOVA), followed by Tukey’s multiple comparison test. We tested the
normal distribution of the data using four methods: Anderson-Darling
test, D’Agostino&Pearson test, Shapiro-Wilk test and Kolmogorov-
Smirnov test. Statistical significance was defined as * (p < 0.05), **
(p < 0.01), or ***(p < 0.001). All analyses were performed using the
software GraphPad Prism v8.0. Pairwise differences in bioluminescent
tumors and T-cell signals were analyzed at selected time points using
Wilcoxon rank-sum test, and the survival data were analyzed using the
Log-rank test.
3. Results
3.1. LNPs programming T cells in vitro
We prepared LNPs encapsulated with CAR plasmid and modified
with CD3 antibody as the targeting ligand on the surface to target T cells.
CD3 antibody was successfully conjugated with phospholipid (Fig. S5).
The nanoparticles were spheroidal as shown in TEM images, with a
particle size of about 200 nm, PDI < 0.2, and a zeta potential close to 0
mV (Fig. 2 A-C). AntiCD3-LNP/CAR19 + shIL6 was incubated in serum
and its size did not significantly change within 7 days (Fig. S6),
demonstrating its good stability in serum. To determine the ability of the
nanoparticles to target T cells in vitro, the LNPs were incubated with T
cells for 4 h. The T cells took up much more CD3 antibody-modified
LNPs than unmodified nanoparticles (Fig. 2D). We investigated the
endosomal escape of LNPs and found that the AntiCD3-LNP/CAR19 +
shIL6 were internalized into the cells by the lysosomal pathway and
could successfully escape the lysosomes at 4 h in vitro. (Fig. S7). The
detection of T cell transduction efficiency by the LNPs helped to deter­
mine the expression of CAR on the surface of T cells. We found that the
transfection efficiency of LNPs was significantly enhanced after CD3
antibody modification, though the enhancement was not very remark­
able (from 4.57 ± 0.30% to 6.9 ± 0.16%, Fig. 2E-F). We detected CD19
CAR positive% of CAR-T cells produced by LNPs and those by lentivirus
with FACS. The results showed that the CAR positive rate of ΔIL6 CAR-T
was higher than that of LNPs (Fig. S8), which might be because the
transfection efficiency of the lentiviral vector was significantly higher
than that of LNPs. We selected different effector: target ratios (E: T) to
evaluate the ability to kill tumor cells after T cell transduction. The △IL-
6 CAR-T group showed the highest killing ability at the effector: target
ratio of 10: 1. The AntiCD3-LNP/CAR19 + shIL6 group showed potent
cytotoxicity and could kill the target cells effectively at E:T = 10:1 and
8:1. The AntiCD3-LNP/CAR19 + shIL6 group showed significantly
enhanced killing effect compared with LNP/CAR19 + shIL6 group at E:
T = 10:1. The AntiCD3-LNP/CAR19 + shIL6 group and LNP/CAR19 +
shIL6 group both showed weak killing ability at E:T = 5: 1 and 1:1
(Fig. 2G). We also examined the ability of T cells to express IL-6 after
killing the target cells. The IL-6 mRNA levels generated in the AntiCD3-
LNP/CAR19 group were significantly higher than those generated in the
LNP/CAR19 + shIL6 and AntiCD3-LNP/CAR19 + shIL6 groups
(Fig. 2H), indicating that IL-6 shRNA in the CAR plasmid effectively
silenced IL-6 expression.

Fig. 2. The LNPs transfected T cells in vitro and produced CAR-T cells. A. The AntiCD3-LNP/CAR19 + shIL6 nanoparticles showed a spheroidal structure when
analyzed by TEM. B and C. The particle size of LNP was about 200 nm, and the zeta potential of LNP was nearly 0 mV. The data are represented as the mean ± SD (n
= 6). D. Confocal microscopy confirmed that the LNPs loaded with DiO dye were internalized by T cells within 4 h (This graph is representative of 3 human donors).
F. The efficiency of T cell transduction with AntiCD3-LNP/CAR19 + shIL6 was significantly higher than that with LNP/CAR19 + shIL6 in vitro. The data are
represented as the mean ± SD of three independent experiments. G. The killing ability of LNPs after T cell transduction following stimulation by K562 cells or Raji
cells. Data are represented as the mean ± SD for 3 technical triplicates, with three random fields of view assessed per replicate. The killing ability was highest at E:T
= 10:1. H. Detection of IL-6 mRNA produced by T-cell programming via qPCR. Data are represented as the mean ± SD of 5 technical triplicates. (The symbol *
represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001).

301
J.-e. Zhou et al. Journal of Controlled Release 350 (2022) 298–307

3.2. LNPs targeting T-cells and producing CAR-T cells In vivo
We used the LNPs to target T lymphocytes in vivo and to convert it
into CAR-T cells. The targeting ability and transduction efficiency of
nanoparticles to T cells in vivo are crucial. First, NOD-SCID IL-2 receptor
gamma null (NSG) mice were injected with Raji-Luc cells through the
tail vein. After seven days, the fluorescence intensity of luciferase was
determined by the small animal in vivo imaging to confirm the leukemia
model. Then, the tumor-bearing mice were randomly placed into one of
four groups, administered with T cells + CD3 antibody-modified LNPs

Fig. 3. CAR DNA-loaded LNPs programed the T cells and produced CAR-T cells in vivo. A. Representative Flow cytometry of the LNPs that bound to and transfected
the T cells. The LNPs showed the ability to program T cells in vivo. This graph is representative of n = 5 tumor-bearing mice. B. Biodistribution of fluorescent CD3
antibody targeting or non-targeted LNPs 24 h after tail-vein injection. Data are from five mice per treatment condition. The mice on the left were administered LNP/
CAR19 + shIL6, and the mice on the right were administered AntiCD3-LNP/CAR19 + shIL6. The CD3 antibody modified the LNPs targeting the lymphoid tissue in
which the T cells were located. C. The number of CAR-T cells produced by LNPs in vivo. The number of CAR-T cells in T+ AntiCD3-LNP/CAR19 + shIL6 group was
significantly higher than that in other groups on the 21st day. Data are represented as mean ± SD of 5 mice in each group. D. Phenotypic analysis of the LNPs after
transfection of T cells (n = 5, Data are represented as mean ± SD). E. The ratio of CD8+/CD4+ CAR-T cells in the peripheral blood. Large quantities of CD8 + CAR-T
were produced and reached highest on Day21 after LNPs administration. The ratio of CD8+/CD4+ CAR-T cells in T + AntiCD3-LNP/CAR19 + shIL6 group was
significantly more than that in other groups on Day 21.

302
J.-e. Zhou et al. Journal of Controlled Release 350 (2022) 298–307

(AntiCD3-LNP/CAR19 + shIL6), T cells+ LNPs (LNP/CAR19 + shIL6), T
cells +CD3 antibody-modified LNPs without knocking down IL-6
(AntiCD3-LNP/CAR19), and conventional CAR-T cells with knocked
down IL-6 function (△IL-6 CAR-T), respectively. The LNPs were injec­
ted 2 h after the T-cell injection in each group. The dose of T cells
injected was 5 × 106 per mouse, and the dose of LNPs was 5 μg/g (based
on the mass of the CAR plasmid). After the tumor-bearing mice were
injected with human T cells and DiR fluorescent dye-labeled AntiCD3-
LNP/CAR19 + shIL6 and LNP/CAR19 + shIL6, their fluorescence dis­
tribution in vivo was observed by the Small Animal In Vivo Imaging
System. Twenty-four hours after injection, while most of LNP/CAR19 +
shIL6 remained in the liver, AntiCD3-LNP/CAR19 + shIL6 was more
accumulated in the lymph nodes of the mice, (Fig. 3B; LNP/CAR19 +
shIL6 on the left and AntiCD3-LNP/CAR19 + shIL6 on the right), which
was attributed to the CD3 antibody modification of LNPs.
To examine whether the LNPs could program the T cells and produce
CAR-T cells in vivo, we determined the number of CAR-T cells produced
in vivo by flow cytometry on 7, 14, 21, 35, and 90 days after injecting T
cells and nanoparticles. The number of CAR-T cells produced in each
group was plotted as a function of time after the administration. T cells

Fig. 4. AntiCD3-LNPs induced an antitumor effect by programming T cells in vivo. Almost all tumor cells were killed after 41 days of injection of T + AntiD3-LNP/
CAR19 + shIL6, which is similar with the positive control group of conventional△IL-6 CAR-T. The antitumor effect of T + LNP/CAR19 + shIL6 was lower than that
of T+ AntiCD3-LNP/CAR19 + shIL6. The injection of T cells, AntiCD3-LNP/CAR19 + shIL6 or LNP/CAR19 + shIL6 alone could not effectively kill tumor cells.

303
J.-e. Zhou et al. Journal of Controlled Release 350 (2022) 298–307

were stably transfected into CAR-T cells in vivo after injecting T+
AntiCD3-LNP/CAR19 + shIL6 or T+ LNP/CAR19 + shIL6. In the T+
AntiCD3-LNP/CAR19 + shIL6 group, a large number of CAR-T cells
were produced by the nanoparticles within 14–21 days after the treat­
ment (mean 74.6% CAR+ among CD3+ T cells on day 21), but the
number sharply decreased within 35–90 days after killing tumor cells
((mean 62.5% CAR+ among CD3+ T cells on day 14, mean 12.1% CAR+
among CD3 + T cells on day 90). In the T+ LNP/CAR19 + shIL6 group,
some T cells were programmed into CAR-T cells in vivo, but the number
of CAR-T cells was lesser than that in the T+ AntiCD3-LNP/CAR19 +
shIL6 group (Fig. 3A). By contrast, only a few CAR-T cells were produced
in the T+ AntiCD3-LNP/CAR19 group. The number of CAR-T cells was
the highest on the 21st day (Fig. 3C). Additionally, the number of CAR-T
cells in T+ AntiCD3-LNP/CAR19 + shIL6 group was significantly higher
than that in T + LNP/CAR 19 + shIL6 group on the 21st day. This
indicated that modifying the CD3 antibody promoted the targeting of
nanoparticles to T cells. The T cell subtypes that internalized the injected
LNPs were measured by flow cytometry, and the proportion of CD8+/
CD4+CAR-T cells was compared (Fig. S9). Most CAR-T cells were CD8+
CAR-T on days 14 and 21, whereas the proportion of CD8+ CAR-T cells

Fig. 5. LNPs programmed T cells caused antitumor effects and knocked down IL-6 in vivo. A. Quantification of bioluminescence intensity of each tumor-bearing
mouse. Each line represents a change in the bioluminescence intensity over time in each mouse. B. Survival of tumor-bearing mice following therapy, depicted
as Kaplan–Meier curves (n = 10). Statistical significance was calculated using the log-rank Mantel-Cox test; * represents P < 0.05, ** represents P < 0.01, and ***
represents P < 0.001. C. The concentration of IL-6 in peripheral blood after nanoparticle injection. AntiCD3-LNP/CAR19 + shIL6 significantly reduced the con­
centration of IL-6 protein, which helped to reduce cytokine syndrome. Data are represented as mean ± SD of 5 mice in each group, and statistical significance was
calculated with ANOVA with Turkey multiple comparisons. D. The concentration of IL-4 in peripheral blood after administration. E. The IL-2 secretion of △IL-6 CAR-
T group in peripheral blood after administration was significantly higher than that of other groups on Day10. F. The secretion of IFN-γ in T + AntiCD3-LNP/CAR19
group, was significantly lower than that of △IL-6 CAR-T, LNP/CAR19 + shIL6, and AntiCD3-LNP/CAR19 + shIL6 groups. The concentration of IFN-γ in T +
AntiCD3-LNP/CAR19 + shIL6 group was significantly higher than that of in T + AntiCD3-LNP/CAR19 group on Day10. G. The concentration of IL-10 in peripheral
blood after administration. T + AntiCD3-LNP/CAR19 group secreted the highest level of IL-10 on Day10. H. The concentration of IL-17α in peripheral blood after
administration. I. The concentration of TNF-α in peripheral blood after administration. T + AntiCD3-LNP/CAR19 group secreted the highest level of TNF-α on Day10.

304
J.-e. Zhou et al.
gradually decreased after the tumor was killed (Fig. 3D). We compared
the CD8+/CD4+ CAR-T cells ratio in different groups on Day 21. The
results showed that the number of CD8 + CAR-T cells in T + AntiCD3-
LNP/CAR19 + shIL6 group was significantly more than that in other
groups on Day 21 (Fig. 3E), indicating that AntiCD3-LNP/CAR19 +
shIL6 could produce more cytotoxic T cells than other types of LNPs.
3.3. LNPs induced an antitumor effect and reduced CRS by programming
T cells
To investigate whether LNPs exerted antitumor effects after T cell
programming in vivo, we systemically injected luciferase-expressing
Raji leukemia cells (Raji-Luc) into NSG mice and used bioluminescent
imaging to assess the differences of tumor burden among treatment
groups (Fig. 4). To compare the therapeutic efficacy of LNPs with con­
ventional CAR T-cell therapy, we set a positive control group of mice
treated with a single dose of 5 × 106 CAR-T cells, which was prepared by
transduction ex vivo with lentiviral vectors encoding CD19 CAR+shIL-6
(△IL-6 CAR-T). We found that almost all tumor cells were killed after
41 days of injection of human T cells + AntiD3-LNP/CAR19 + shIL6,
which is similar to the positive control group of conventional CAR T-cell
therapy. This could be because T cells are programmed by AntiCD3-
LNP/CAR19 + shIL6, and thus, transformed into CAR-T cells in vivo
(Fig. 3C) that show strong antitumor effects comparable to conventional
CAR-T therapy. T cells + LNP/CAR19 + shIL6 also produced antitumor
effect in mice, which, however, was lower than that of T+ AntiCD3-
LNP/CAR19 + shIL6. This is probably because LNP/CAR19 + shIL6
could not efficiently program T cells in vivo, leading to producing an
inadequate number of CAR-T cells. Moreover, the injection of human T
cells alone could not kill tumor cells in mice. Similarly, the injection of
AntiCD3-LNP/CAR19 + shIL6 or LNP/CAR19 + shIL6 alone could not
kill tumor cells, which might be due to the lack of T cells in NSG mice
and the failure of LNPs to program T cells to express CAR. We also found
that T cells + AntiCD3-LNP/CAR19 showed a slight antitumor effect in
vivo, which was lower than that of T cells + AntiCD3-LNP/CAR19 +
shIL6. This was mainly due to the low encapsulation efficiency of
AntiCD3-LNP/CAR19, which was significantly lower than AntiCD3-
LNP/CAR19 + shIL6 (Table S2), thus causing poor transduction of T
cells and fewer production of CAR-T cells in vivo (Fig. 3C).
Fluorescence intensity analysis was performed on five representative
mice from each group (Fig. 5A). We found that T cells +AntiCD3-LNP/
CAR19 + shIL6 could reduce tumor burden from day 12, indicating that
nanoparticles exhibit an antitumor effect by programming T cells in
vivo. We evaluated the survival time of tumor-bearing mice in each
group for 120 days after administration (Fig. 5B). We found that mice
treated with T + AntiCD3-LNP/CAR19 + shIL6 survived significantly
longer than those treated with T+ LNP/CAR19 + shIL6. The therapeutic
effect of T + AntiCD3-LNP/CAR19 + shIL6 was not significantly
different from that of △IL-6 CAR-T cells. The median survival times of
mice injected with PBS, human T cells alone, LNP/CAR19 + shIL6,
AntiCD3-LNP/CAR19 + shIL6, and T + AntiCD3-LNP/GFP were 20, 19,
12, 13, and 31 days, respectively. These results indicated that the in­
jection of human T cells or LNP/CAR19 + shIL6 or AntiCD3-LNP/
CAR19 + shIL6 alone cannot significantly prolong the survival of tumor-
bearing mice. To determine whether CRS is produced in tumor-bearing
mice after administration, we collected the serum from the mice on days
7, 10, and 14 after administration and detected the concentration of
seven types of cytokines. After injection of human T cells+ AntiCD3-
LNP/CAR19, tumor-bearing mice produced high levels of IL-6 on the
10th and 14th day (Fig. 5C), which was significantly higher than that in
T + AntiCD3-LNP/CAR19 + shIL6 group. The results indicated that
AntiCD3-LNP/CAR19 + shIL6 transfected T cells successfully and
knocked down IL-6 in vivo, which is helpful to reduce the incidence and
degree of CRS. We found that the secretion of IFN-γ which related to
tumor killing in T + AntiCD3-LNP/CAR19 group, was significantly
lower than that of △IL-6 CAR-T, LNP/CAR19 + shIL6, and AntiCD3-
305
Journal of Controlled Release 350 (2022) 298–307
LNP/CAR19 + shIL6 groups. This might be the main reason for the low
survival rate of mice in T + AntiCD3-LNP/CAR19 group. Tumor
microenvironment usually has high levels of IL-4 [18], which promotes
immunosuppression and the production of pro-inflammatory cytokines.
IL-17A can promote the activation of T cells and enhance the recruit­
ment of autoimmune cells in patients. There was no significant differ­
ence of the two cytokines among different groups (Fig. 5D, Fig. 5H). IL-
2, IL-10, and TNF-α are pro-inflammatory cytokines that can activate a
series of inflammatory-related cascades, which are closely related to the
occurrence of CRS. T + AntiCD3-LNP/CAR19 + shIL6 group produced
lower levels of pro-inflammatory factors than T + AntiCD3-LNP/CAR19
group on Day10 (Fig. 5E, G, and I), which is helpful to reduce the
incidence of CRS.
4. Discussion
The traditional CAR-T cells need to be prepared ex vivo, which has
several problems, such as a complex production process, long cycle,
strict quality control requirements, personal customization, and high
cost. Moreover, the problem of cytokine release syndrome (CRS) is
common in the clinical application of CAR-T. To solve these problems, in
this study, we used a mixture of ionizable phospholipids DLin-MC3-DMA
[32,33] and cationic lipids [34–36] as the material to prepare lipid
nanoparticles and modified the CD3 antibody on the surface so that it
could be actively targeted to and internalized by T cells. The combined
plasmids containing IL-6 shRNA and CD19-CAR loaded by LNPs stably
transfected T cells under the influence of the nuclear localization peptide
MTAS-NLS to produce IL-6 knockdown CAR-T cells. Therefore, the
technology could avoid complex preparation process ex vivo and in­
crease the convenience of CAR-T therapy; At the same time, it could
effectively reduce the CRS problem associated with CAR-T therapy and
improve its clinical safety.
The rational design of gene delivery system is the key factor influ­
encing the effective delivery and transfection of genes in vivo. The safety
of LNPs editing T cells in vivo determines whether they can be used
clinically. The component of phospholipid used for LNPs is referenced
from ONPATTRO [37] and the Selective organ targeting (SORT) nano­
particles [38]. We used cationic lipid DOTAP and ionizable cationic lipid
DLinDMA as the main carrier materials of LNPs [39]. DLin-MC3-DMA
has a neutral charge at around pH 7.0, which keeps LNPs stable in the
blood, and its positive charge at low pH enables LNPs to escape from
endosomes. Therefore, ionizable cationic lipids have shown advantages
in nucleic acid delivery in vivo [40]. Currently, EndoTAG-1 [41,42], a
paclitaxel cationic liposome prepared from DOTAP, has entered clinical
phase III research. However, a high positive charge of LNPs might cause
serious immune reactions and toxic side effects on normal tissues
[43–45]. Therefore, maintaining a low or neutral charge is very
important for LNPs. We optimized the formulation based on previous
siRNA-loaded LNPs [32,40,46], so that the zeta potential of LNP was
maintained at 0–2 mV by adjusting the amounts of pDNA, DOTAP, and
Dlin-MC3-DMA (Table S1).
The transfection efficiency of LNP depends on the size of the pDNA
and makes it easier for smaller pDNA copies [47,48]. Smaller DNA
fragments are considered to be more easily encapsulated or more easily
translocated to the nucleus. To ensure the high transfection efficiency of
AntiCD3-LNP/CAR19 + shIL6 in vivo, we added IL-6 shRNA based on
the original CAR19 pDNA and deleted many unnecessary components,
thus reducing the copies of the plasmid (5314 bp; lesser than CAR19
(8504 bp) in AntiCD3-LNP/CAR 19 group) to facilitate the lipid
encapsulation (Fig. S1 and S2); In addition, we synthesized microtubule-
associated nuclear localization peptide (MTAS-NLS)-DSPE (Fig. S3,
Fig. S4) and added it into LNPs, promoting LNPs to enter the nucleus
while simultaneously adsorbing and compressing the plasmid. The main
reason is that delivering large DNA sequences need more positive
charges (high N/P ratio) to increase DNA condensation within the
nanoparticles. We encapsulated AntiCD3-LNPs/CAR19 with the same
J.-e. Zhou et al.
amount of positive charge as AntiCD3-LNPs/CAR19 + shIL6, whereas
the size of CAR19 was much larger than that of CAR19 + shIL6, thus
resulting in insufficient condensation of CAR19 and low encapsulation
efficiency (Table S2). In addition, AntiCD3-LNP/CAR19 has more
negative charges (− 3.4 ± 0.1). These could be the reasons for poor T cell
transduction, CAR-T cell production, and antitumor effect in the
AntiCD3-LNP/CAR19 group. The original transposon iPB7 was inserted
in the plasmid for stable transfection of T cells by LNPs, thus T cells were
able to stable express CAR gene in vivo. The results showed that it could
last for >90 days after T cell transduction.
We successfully programmed T cells in vivo, but the off-target effect
during treatment may reduce the safety. We found that some LNPs
transfected macrophages in vivo, and we also examined the number of
macrophages that expressed CAR (Fig. S10). A small portion of macro­
phages was transfected and expressed CAR, but these CAR-macrophages
were difficult to proliferate in vivo. By contrast, the CAR-T cells pro­
duced in vivo could continuously proliferate under the stimulation of
tumor cells. No significant toxicity was observed through hematoxylin
and eosin (H&E) staining of sections of major organs (Fig. S11). This
indicated that the expression of CAR in non-T cells did not cause sig­
nificant damage to other organs.
In conclusion, we found that the in vivo programming of T cells into
CAR-T cells via LNPs achieved antitumor effect comparable to that of
conventionally CAR-T therapy and reduced CRS risk by knocking down
IL-6. This technology improves the convenience of using CAR-T tech­
nology, and is helpful to further promote the clinical application.
Funding
This work was supported by the National Natural Science Foundation
of China (81872812, 82073800).
Credit author statement
Jing-e Zhou: Perform experiments, analyze data, and writing the
manuscript.
Lei Sun: Perform experiments, analyze data, and writing the
manuscript.
Yujie Jia: Perform experiment.
Zhehao Wang:Perform experiment.
Tengshuo Luo: Perform experiments.
Jingwen Tan: Perform experiments.
Xiaoyan Fang:Perform experiments.
Hongjia Zhu:Perform experiments.
Jing Wang:Support the program and review the manuscript.
Lei Yu: Design and support the program and review the manuscript.
Zhiqiang Yan: Design and support the program, writing and review
the manuscript.
Declaration of Competing Interest
The authors have no competing interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jconrel.2022.08.033.
References
[1] FDA approves second CAR T-cell therapy, Cancer Discov. 8 (2018) 5–6.
[2] T.J. Fry, N.N. Shah, R.J. Orentas, M. Stetler-Stevenson, C.M. Yuan, S. Ramakrishna,
P. Wolters, S. Martin, C. Delbrook, B. Yates, H. Shalabi, T.J. Fountaine, J.F. Shern,
R.G. Majzner, D.F. Stroncek, M. Sabatino, Y. Feng, D.S. Dimitrov, L. Zhang,
S. Nguyen, H. Qin, B. Dropulic, D.W. Lee, C.L. Mackall, CD22-targeted CAR T cells
induce remission in B-ALL that is naive or resistant to CD19-targeted CAR
immunotherapy, Nat. Med. 24 (2018) 20–28.
306
Journal of Controlled Release 350 (2022) 298–307
[3] C. Sheridan, First approval in sight for Novartis’ CAR-T therapy after panel vote,
Nat. Biotechnol. 35 (2017) 691–693.
[4] J.N. Brudno, R.P. Somerville, V. Shi, J.J. Rose, D.C. Halverson, D.H. Fowler, J.
C. Gea-Banacloche, S.Z. Pavletic, D.D. Hickstein, T.L. Lu, S.A. Feldman, A.
T. Iwamoto, R. Kurlander, I. Maric, A. Goy, B.G. Hansen, J.S. Wilder, B. Blacklock-
Schuver, F.T. Hakim, S.A. Rosenberg, R.E. Gress, J.N. Kochenderfer, Allogeneic T
cells that express an anti-CD19 chimeric antigen receptor induce remissions of B-
cell malignancies that Progress after allogeneic hematopoietic stem-cell
transplantation without causing graft-versus-host disease, J. Clin. Oncol. 34 (2016)
1112–1121.
[5] J.A. Fraietta, K.A. Beckwith, P.R. Patel, M. Ruella, Z. Zheng, D.M. Barrett, S.
F. Lacey, J.J. Melenhorst, S.E. McGettigan, D.R. Cook, C. Zhang, J. Xu, P. Do,
J. Hulitt, S.B. Kudchodkar, A.P. Cogdill, S. Gill, D.L. Porter, J.A. Woyach, M. Long,
A.J. Johnson, K. Maddocks, N. Muthusamy, B.L. Levine, C.H. June, J.C. Byrd, M.
V. Maus, Ibrutinib enhances chimeric antigen receptor T-cell engraftment and
efficacy in leukemia, Blood 127 (2016) 1117–1127.
[6] P. Kebriaei, H. Singh, M.H. Huls, M.J. Figliola, R. Bassett, S. Olivares, B. Jena, M.
J. Dawson, P.R. Kumaresan, S. Su, S. Maiti, J. Dai, B. Moriarity, M.A. Forget,
V. Senyukov, A. Orozco, T. Liu, J. McCarty, R.N. Jackson, J.S. Moyes, G. Rondon,
M. Qazilbash, S. Ciurea, A. Alousi, Y. Nieto, K. Rezvani, D. Marin, U. Popat,
C. Hosing, E.J. Shpall, H. Kantarjian, M. Keating, W. Wierda, K.A. Do, D.
A. Largaespada, D.A. Lee, P.B. Hackett, R.E. Champlin, L.J. Cooper, Phase I trials
using sleeping beauty to generate CD19-specific CAR T cells, J. Clin. Invest. 126
(2016) 3363–3376.
[7] C.J. Turtle, L.A. Hanafi, C. Berger, T.A. Gooley, S. Cherian, M. Hudecek,
D. Sommermeyer, K. Melville, B. Pender, T.M. Budiarto, E. Robinson, N.
N. Steevens, C. Chaney, L. Soma, X. Chen, C. Yeung, B. Wood, D. Li, J. Cao,
S. Heimfeld, M.C. Jensen, S.R. Riddell, D.G. Maloney, CD19 CAR-T cells of defined
CD4+:CD8+ composition in adult B cell ALL patients, J. Clin. Invest. 126 (2016)
2123–2138.
[8] R.C. Sterner, R.M. Sterner, CAR-T cell therapy: current limitations and potential
strategies, Blood Cancer J. 11 (2021) 69.
[9] I.R. Marco, L. Davila, Xiuyan Wang, Shirley Bartido, Jae Park, Kevin Curran,
Stephen S. Chung, Jolanta Stefanski, Oriana Borquez-Ojeda,
Malgorzata Olszewska, Jinrong Qu, Teresa Wasielewska, Qing He, Mitsu Fink,
Himaly Shinglot, Maher Youssif, Mark Satter, Yongzeng Wang, James Hosey,
Hilda Quintanilla, Elizabeth Halton, Yvette Bernal, Diana C.G. Bouhassira, Maria
E. Arcila, Mithat Gonen, Gail J. Roboz, Peter Maslak, Dan Douer, Mark G. Frattini,
Sergio Giralt Michel Sadelain, Renier Brentjens, Efficacy and toxicity management
of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia, Cancer 6
(2014).
[10] B.L. Levine, J. Miskin, K. Wonnacott, C. Keir, Global manufacturing of CAR T cell
therapy, Mol. Ther. Methods Clin. Dev. 4 (2017) 92–101.
[11] X. Wang, I. Riviere, Clinical manufacturing of CAR T cells: foundation of a
promising therapy, Mol. Ther. Oncolytics 3 (2016) 16015.
[12] S.S. Neelapu, S. Tummala, P. Kebriaei, W. Wierda, C. Gutierrez, F.L. Locke, K.
V. Komanduri, Y. Lin, N. Jain, N. Daver, J. Westin, A.M. Gulbis, M.E. Loghin, J.
F. de Groot, S. Adkins, S.E. Davis, K. Rezvani, P. Hwu, E.J. Shpall, Chimeric antigen
receptor T-cell therapy - assessment and management of toxicities, Nat. Rev. Clin.
Oncol. 15 (2018) 47–62.
[13] S.S. Neelapu, S. Tummala, P. Kebriaei, W. Wierda, F.L. Locke, Y. Lin, N. Jain,
N. Daver, A.M. Gulbis, S. Adkins, K. Rezvani, P. Hwu, E.J. Shpall, Toxicity
management after chimeric antigen receptor T cell therapy: one size does not fit
‘ALL’, Nat. Rev. Clin. Oncol. 15 (2018) 218.
[14] S.L. Maude, N. Frey, P.A. Shaw, R. Aplenc, D.M. Barrett, N.J. Bunin, A. Chew, V.
E. Gonzalez, Z. Zheng, S.F. Lacey, Y.D. Mahnke, J.J. Melenhorst, S.R. Rheingold,
A. Shen, D.T. Teachey, B.L. Levine, C.H. June, D.L. Porter, S.A. Grupp, Chimeric
antigen receptor T cells for sustained remissions in leukemia, N. Engl. J. Med. 371
(2014) 1507–1517.
[15] F.L. Locke, S.S. Neelapu, N.L. Bartlett, T. Siddiqi, J.C. Chavez, C.M. Hosing,
A. Ghobadi, L.E. Budde, A. Bot, J.M. Rossi, Y. Jiang, A.X. Xue, M. Elias, J. Aycock,
J. Wiezorek, W.Y. Go, Phase 1 results of ZUMA-1: a multicenter study of KTE-C19
anti-CD19 CAR T cell therapy in refractory aggressive lymphoma, Mol. Ther. 25
(2017) 285–295.
[16] R.Q. Le, L. Li, W. Yuan, S.S. Shord, L. Nie, B.A. Habtemariam, D. Przepiorka, A.
T. Farrell, R. Pazdur, FDA approval summary: tocilizumab for treatment of
chimeric antigen receptor T cell-induced severe or life-threatening cytokine release
syndrome, Oncologist 23 (2018) 943–947.
[17] L. Kang, X. Tang, J. Zhang, M. Li, N. Xu, W. Qi, J. Tan, X. Lou, Z. Yu, J. Sun,
Z. Wang, H. Dai, J. Chen, G. Lin, D. Wu, L. Yu, Interleukin-6-knockdown of
chimeric antigen receptor-modified T cells significantly reduces IL-6 release from
monocytes, Exp. Hematol. Oncol. 9 (2020) 11.
[18] T. Jain, M. Bar, A.J. Kansagra, E.A. Chong, S.K. Hashmi, S.S. Neelapu, M. Byrne,
E. Jacoby, A. Lazaryan, C.A. Jacobson, S.M. Ansell, F.T. Awan, L. Burns,
V. Bachanova, C.M. Bollard, P.A. Carpenter, J.F. DiPersio, M. Hamadani, H.
E. Heslop, J.A. Hill, K.V. Komanduri, C.A. Kovitz, H.M. Lazarus, J.M. Serrette,
M. Mohty, D. Miklos, A. Nagler, S.Z. Pavletic, B.N. Savani, S.J. Schuster, M.
A. Kharfan-Dabaja, M.A. Perales, Y. Lin, Use of chimeric antigen receptor T cell
therapy in clinical practice for relapsed/refractory aggressive B cell non-Hodgkin
lymphoma: an expert panel opinion from the American Society for Transplantation
and Cellular Therapy, Biol. Blood Marrow Transpl. 25 (2019) 2305–2321.
[19] F. Bonacina, D. Coe, G. Wang, M.P. Longhi, A. Baragetti, A. Moregola,
K. Garlaschelli, P. Uboldi, F. Pellegatta, L. Grigore, L. Da Dalt, A. Annoni,
S. Gregori, Q. Xiao, D. Caruso, N. Mitro, A.L. Catapano, F.M. Marelli-Berg, G.
D. Norata, Myeloid apolipoprotein E controls dendritic cell antigen presentation
and T cell activation, Nat. Commun. 9 (2018) 3083.
J.-e. Zhou et al.
[20] E. Roselli, R. Faramand, M.L. Davila, Insight into next-generation CAR
therapeutics: designing CAR T cells to improve clinical outcomes, J. Clin. Invest.
131 (2021).
[21] N.N. Parayath, M.T. Stephan, In situ programming of CAR T cells, Annu. Rev.
Biomed. Eng. 23 (2021) 385–405.
[22] A.R.K. Kumar, Y. Shou, B. Chan, K.L.A. Tay, Materials for improving immune cell
transfection, Adv. Mater. 33 (2021), e2007421.
[23] M.M. Billingsley, A.G. Hamilton, D. Mai, S.K. Patel, K.L. Swingle, N.C. Sheppard, C.
H. June, M.J. Mitchell, Orthogonal Design of Experiments for optimization of lipid
nanoparticles for mRNA engineering of CAR T cells, Nano Lett. 22 (1) (2021)
533–542.
[24] T.T. Smith, S.B. Stephan, H.F. Moffett, L.E. McKnight, W. Ji, D. Reiman,
E. Bonagofski, M.E. Wohlfahrt, S.P.S. Pillai, M.T. Stephan, In situ programming of
leukaemia-specific T cells using synthetic DNA nanocarriers, Nat. Nanotechnol. 12
(2017) 813–820.
[25] X. Han, Y. Wang, W.D. Han, Chimeric antigen receptor modified T-cells for cancer
treatment, Chronic. Dis. Transl. Med. 4 (2018) 225–243.
[26] R. Ballotti, Y. Cheli, C. Bertolotto, The complex relationship between MITF and the
immune system: a melanoma ImmunoTherapy (response) factor? Mol. Cancer 19
(2020) 170.
[27] N.N. Parayath, S.B. Stephan, A.L. Koehne, P.S. Nelson, M.T. Stephan, In vitro-
transcribed antigen receptor mRNA nanocarriers for transient expression in
circulating T cells in vivo, Nat. Commun. 11 (2020) 6080.
[28] K. Narayanan, S.K. Yen, Q. Dou, P. Padmanabhan, T. Sudhaharan, S. Ahmed, J.
Y. Ying, S.T. Selvan, Mimicking cellular transport mechanism in stem cells through
endosomal escape of new peptide-coated quantum dots, Sci. Rep. 3 (2013) 2184.
[29] K. Kogure, R. Moriguchi, K. Sasaki, M. Ueno, S. Futaki, H. Harashima, Development
of a non-viral multifunctional envelope-type nano device by a novel lipid film
hydration method, J. Control. Release 98 (2004) 317–323.
[30] J.A. Kulkarni, D. Witzigmann, J. Leung, Y.Y.C. Tam, P.R. Cullis, On the role of
helper lipids in lipid nanoparticle formulations of siRNA, Nanoscale 11 (2019)
21733–21739.
[31] E. Ambegia, S. Ansell, P. Cullis, J. Heyes, L. Palmer, I. MacLachlan, Stabilized
plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation
lifetimes and tumor selective gene expression, Biochim. Biophys. Acta 1669 (2005)
155–163.
[32] J.J. Wheeler, L.P.M. Ossanlou, P.R. Cullis, Stabilized plasmid-lipid particles:
construction and characterization, Gene Ther. 6 (1999) 271–281.
[33] S.C. Semple, A. Akinc, J. Chen, A.P. Sandhu, B.L. Mui, C.K. Cho, D.W. Sah,
D. Stebbing, E.J. Crosley, E. Yaworski, I.M. Hafez, J.R. Dorkin, J. Qin, K. Lam, K.
G. Rajeev, K.F. Wong, L.B. Jeffs, L. Nechev, M.L. Eisenhardt, M. Jayaraman,
M. Kazem, M.A. Maier, M. Srinivasulu, M.J. Weinstein, Q. Chen, R. Alvarez, S.
A. Barros, S. De, S.K. Klimuk, T. Borland, V. Kosovrasti, W.L. Cantley, Y.K. Tam,
M. Manoharan, M.A. Ciufolini, M.A. Tracy, A. de Fougerolles, I. MacLachlan, P.
R. Cullis, T.D. Madden, M.J. Hope, Rational design of cationic lipids for siRNA
delivery, Nat. Biotechnol. 28 (2010) 172–176.
[34] N. Skandrani, A. Barras, D. Legrand, T. Gharbi, H. Boulahdour, R. Boukherroub,
Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and
drug co-delivery and cell imaging, Nanoscale 6 (2014) 7379–7390.
[35] L. Zhang, P. Wang, Q. Feng, N. Wang, Z. Chen, Y. Huang, W. Zheng, X. Jiang, Lipid
nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy, NPG
Asia Mater. 9 (2017) e441.
307
Journal of Controlled Release 350 (2022) 298–307
[36] J. Buck, P. Grossen, P.R. Cullis, J. Huwyler, D. Witzigmann, Lipid-based DNA
therapeutics: hallmarks of non-viral gene delivery, ACS Nano 13 (2019)
3754–3782.
[37] A. Akinc, M.A. Maier, M. Manoharan, K. Fitzgerald, M. Jayaraman, S. Barros,
S. Ansell, X. Du, M.J. Hope, T.D. Madden, B.L. Mui, S.C. Semple, Y.K. Tam,
M. Ciufolini, D. Witzigmann, J.A. Kulkarni, R. van der Meel, P.R. Cullis, The
Onpattro story and the clinical translation of nanomedicines containing nucleic
acid-based drugs, Nat. Nanotechnol. 14 (2019) 1084–1087.
[38] L.T.J. Qiang Cheng, J. Daniel, Tuo Wei Siegwart, Lukas Farbiak, Sean A. Dilliard,
Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery
and CRISPR–Cas gene editing, Nat. Nanotechnol. 15 (2020) 313–320.
[39] P. de Antonellis, L. Liguori, A. Falanga, M. Carotenuto, V. Ferrucci, I. Andolfo,
F. Marinaro, I. Scognamiglio, A. Virgilio, G. De Rosa, A. Galeone, S. Galdiero,
M. Zollo, MicroRNA 199b-5p delivery through stable nucleic acid lipid particles
(SNALPs) in tumorigenic cell lines, Naunyn Schmiedeberg’s Arch. Pharmacol. 386
(2013) 287–302.
[40] D. Adams, A. Gonzalez-Duarte, W.D. O’Riordan, C.C. Yang, M. Ueda, A.V. Kristen,
I. Tournev, H.H. Schmidt, T. Coelho, J.L. Berk, K.P. Lin, G. Vita, S. Attarian,
V. Plante-Bordeneuve, M.M. Mezei, J.M. Campistol, J. Buades, T.H. Brannagan 3rd,
B.J. Kim, J. Oh, Y. Parman, Y. Sekijima, P.N. Hawkins, S.D. Solomon,
M. Polydefkis, P.J. Dyck, P.J. Gandhi, S. Goyal, J. Chen, A.L. Strahs, S.V. Nochur,
M.T. Sweetser, P.P. Garg, A.K. Vaishnaw, J.A. Gollob, O.B. Suhr, Patisiran, an RNAi
therapeutic, for hereditary transthyretin amyloidosis, N. Engl. J. Med. 379 (2018)
11–21.
[41] A. Awada, I.N. Bondarenko, J. Bonneterre, E. Nowara, J.M. Ferrero, A.V. Bakshi,
C. Wilke, M. Piccart, C.T.s. group, a randomized controlled phase II trial of a novel
composition of paclitaxel embedded into neutral and cationic lipids targeting
tumor endothelial cells in advanced triple-negative breast cancer (TNBC), Ann.
Oncol. 25 (2014) 824–831.
[42] U. Fasol, A. Frost, M. Buchert, J. Arends, U. Fiedler, D. Scharr, J. Scheuenpflug,
K. Mross, Vascular and pharmacokinetic effects of EndoTAG-1 in patients with
advanced cancer and liver metastasis, Ann. Oncol. 23 (2012) 1030–1036.
[43] H. Lv, S. Zhang, B. Wang, S. Cui, J. Yan, Toxicity of cationic lipids and cationic
polymers in gene delivery, J. Control. Release 114 (2006) 100–109.
[44] S. Daniel, A.C. Friend, J. Robert, C. Debs, Endocytosis and intracellular processing
accompanying transfection mediated by cationic liposomes, Biochim. Biophys.
Acta 1278 (1996) 41–50.
[45] D. Mirska, K. Schirmer, S.S. Funari, A. Langner, B. Dobner, G. Brezesinski,
Biophysical and biochemical properties of a binary lipid mixture for DNA
transfection, Colloids Surf. B: Biointerfaces 40 (2005) 51–59.
[46] S.M.A. Muthusamy Jayaraman, Barbara L. Mui, Ying K. Tam, Jianxin Chen,
Xinyao Du, David Butler, Laxman Eltepu, Shigeo Matsuda, Jayaprakash
K. Narayanannair, Kallanthottathil G. Rajeev, Ismail M. Hafez, Akin Akinc, Martin
A. Maier, Mark A. Tracy, Pieter R. Cullis, Thomas D. Madden, Muthiah Manoharan,
J. Michael, Hope, maximizing the potency of siRNA lipid nanoparticles for hepatic
gene silencing in vivo, Angew. Chem. Int. Ed. 51 (2012) 8529.
[47] K. Remaut, N.N. Sanders, F. Fayazpour, J. Demeester, S.C. De Smedt, Influence of
plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes,
J. Control. Release 115 (2006) 335–343.
[48] M. Brgles, M. Santak, B. Halassy, D. Forcic, J. Tomasic, Influence of charge ratio of
liposome/DNA complexes on their size after extrusion and transfection efficiency,
Int. J. Nanomedicine 7 (2012) 393–401.