JID: ENGREG

ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

Engineered Regeneration xxx (xxxx) xxx

Contents lists available at ScienceDirect

Engineered Regeneration
journal homepage: http://www.keaipublishing.com/en/journals/engineered-regeneration/

Microalgae-based drug delivery systems in biomedical applications

Hui Huang a,b,f, Yutong Lang d,f, Shoujie Wang c,∗∗, Min Zhou a,c,d,e,f,∗
a
Eye Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310029, China
b
Institute of Translational Medicine, Zhejiang University, Hangzhou 310029, China
c
Plastic Surgery, The Fouthe Affiliated Hospital, Zhejiang University School of Medicine, Yiwu 322000, China
d
Zhejiang University-University of Edinburgh Institute (ZJU-UoE Institute), Zhejiang University, Haining 314400, China
e
The National Key Laboratory of Biobased Transportation Fuel Technology, Zhejiang University, Hangzhou 310027, China
f
ZJU-Etuoke Joint Research Centre, Zhejiang University, Haining, 310029, China

a r t i c l e i n f o a b s t r a c t

Keywords: Over decades of development, the modern drug delivery system continues to grapple with numerous challenges,
Microalgae including drug loading inefficiencies, issues of immunogenicity, and cytotoxicity. These limitations restrict its
Drug delivery system application across various systems. Microalgae, as a natural resource, are not only abundant in bioactive com-
Tumor hypoxia
pounds but also possess multiple biological properties, including active surface, photosynthesis capabilities, and
Oxygenator
excellent biocompatibility. These attributes make microalgae highly promising as carriers for targeted drug de-
Microswimmer
livery, offering significant potential for the diagnosis and treatment of various diseases. Therefore, leveraging the
exceptional properties of microalgae for drug delivery and optimizing their qualities is of paramount importance.
This article focuses on elucidating the biological characteristics of microalgae and their applications in drug de-
livery, with a particular emphasis on emerging strategies for efficient drug loading and precise targeted delivery.
Microalgae, as a natural biomaterial, hold immense potential for both commercial and clinical applications.

1. Introduction
In 1950, modern drug delivery technology appeared and attracted
great attention in the field of therapeutics, which has been developed
rapidly during this 60-year period [1]. Drug delivery technologies have
greatly enhanced targeted therapy and increased treatment compliance
of patients, thereby facilitating the successful rate of therapies [2,3].
There are two outstanding issues that must be overcome for modern
drug delivery. The first barrier is the physicochemical problem, such
as poor water solubility of drugs and uncontrolled drug release kinetics.
The other one is the biological problem, the distribution of drug delivery
systems in the organism cannot be controlled by formulation properties
[1]. Therefore, developing smarter materials to overcome these barriers
is an urgent problem in the field of drug delivery. Biomaterials which is
nonpoisonous, biodegradable, highly biocompatible, and naturally syn-
thesized, become auspicious candidates as drug carriers [4–6].
Microalgae are microscopic photosynthetic organisms with lengths
ranging from a few microns to a few hundred microns that can only
be seen under the microscope. Microalgae have a wide living environ-
ment and strong adaptability, which almost exist in all ecosystems [7,8].
These microorganisms have already been widely used in pharmaceu-
tical, cosmetic, food, nutraceutical industries, and biomedicine due to
their bioactive substances, such as proteins, vitamins, and fatty acids
[9–11]. However, in recent years, the biological properties of microal-
gae biomass itself is getting more attentions for multiple usage, such as
photosynthesis, active surface, and good biocompatibility [12,13]. Ac-
cording to these excellent biological properties, microalgae has become
one of the most potential drug delivery systems [12,14].
Microalgae have already been used in biomedicine in the past
decade, with excellent progress. It has been found that microalgae can
strongly adsorb micromolecules, nanoparticles, and various metal ions
through simple ways (electrostatic interactions and physical deposition)
and complex processes (physicochemical and biochemical reactions)
[15]. Consequently, this strong drug adsorption capability of microal-
gae makes them ideal biomaterials to effectively carry anticancer drugs,
antibiotics, and functional nanoparticles with or without artificial mod-
ification. Therefore, microalgae are widely utilized in the domain of
drug delivery, which already showed excellent results [12,16-18]. Other
than adsorption, photosynthesis, high biocompatibility and micron size
of microalgae are also important reasons for them to become ideal drug
carriers [14,19].
Microalgae are widely used in many fields of medicine recently, and
offered new insight into wound healing [20,21], anti-tumor [22–24],
bioanalysis [25–27] and tissue engineering [28–30]. Although the stud-

∗
Corresponding author at: Eye Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310029, China.
∗∗
Corresponding author.
E-mail addresses: Wangshoujie@zju.edu.cn (S. Wang), zhoum@zju.edu.cn (M. Zhou).

https://doi.org/10.1016/j.engreg.2024.01.002
Received 19 November 2023; Received in revised form 12 January 2024; Accepted 12 January 2024
Available online xxx
2666-1381/© 2024 The Authors. Publishing Services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Please cite this article as: H. Huang, Y. Lang, S. Wang et al., Microalgae-based drug delivery systems in biomedical applications, Engineered
Regeneration, https://doi.org/10.1016/j.engreg.2024.01.002
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
ies of microalgae as drug carriers are generally a fledgling field of re-
search, it is promising that the applications of microalgae as drug deliv-
ery systems will be deeply researched in the near future. We also firmly
believe that these natural biomaterials would be translated into clin-
ical practice. This review aims to highlight microalgae applications as
drug delivery systems by systematically analyzing the preclinical studies
(Fig. 1).
2. Applications of microalgae biomass as drug carrier
2.1. Microalgae drug delivery systems based on simple preparation
Conventional drug delivery system often presents some common
shortcomings, such as low bioavailability, limited drug solubility, short
activity, and poor pharmacokinetic profiles [31–33]. However, microal-
gae, with high biocompatibility, low cost, large active surface area, and
high-speed propulsion, are ideal biomaterials for drug delivery to over-
come these disadvantages [12,14]. Microalgae drug delivery systems
could be designed based on their inherent properties of microalgae, such
as active surface. For example, Gnanamoorthy et al. [34] explored the
antibiotic streptomycin loading and releasing from a kind of diatom Cos-
cinodiscus concinnus. Results indicated that the drug could be released
rapidly from the surface of diatom foramen at first 6 h, and released
sustainably from the cribrum and cribellum for up to 7 days. In our
one work, Spirulina platensis is also found to be an ideal biotemplate to
construct a microalgae drug delivery system targeting lung owning to
its charged surface [18]. The positively charged doxorubicin (DOX) can
be loaded to the negatively charged surface of S. platensis through non-
covalent electrostatic interactions. This DOX-loaded S. platensis system
2
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
Fig. 1. Design of microalgae-based drug deliv-
ery systems and their diverse biomedical appli-
cations.
(SP@DOX) is easy to be captured by pulmonary capillaries and pas-
sively targeted to the lung because of its special spiral shape and mi-
cron size. SP@DOX showed excellent targeted therapeutic ability in the
4T1 breast cancer lung metastasis mouse model through intravenous
injection (Fig. 2(A)–(D)). Curcumin is also a positively charged drug,
which can be efficiently loaded to S. platensis via noncovalent elec-
trostatic interactions, in our another work, we designed the drug de-
livery SP@Curcumin to treat colon cancer and colitis [17]. In those
two models, SP@Curcumin could inhibit tumor progression in combi-
nation with chemo- and radio-therapy, and protect healthy tissues by
removing reactive oxygen species (ROS) induced by X-ray radiation.
Moreover, S. platensis can also load drugs utilizing its natural junctional
pores and aqueous channels on the cell wall. Amifostine was success-
fully loaded into S. platensis cells by our team to fabricate oral microcar-
riers (SP@AMF) for intestinal protection in cancer radiotherapy [16].
SP@AMF has a superior performance of drug distribution and bioavail-
ability than free drug and its enteric capsule, and it can effectively pre-
vent radiation-induced intestine injury and maintain intestinal micro-
biota homeostasis (Fig. 2(E)–(G)). Owning to the simple preparation of
microalgae drug delivery system based on microalgae characteristics it-
self, microalgae has great protential in the commercial and clinical set-
ting.
2.2. Microalgae drug delivery systems based on surface modifying
However, some microalgae can hardly carry specific drugs or have
very low drug loading efficiency. Recently, more and more researchers
focus on constructing smart drug delivery systems with higher drug-
loading efficiency and multifunctionality by modifying microalgae.
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 2. Microalgae drug delivery system based on simple preparation. (A) The schematic illustration of drug delivery system based on SP@DOX targeting lung and
inhibition of metastasis of breast cancer to lung. (B) In vitro chemotherapeutic effect of SP@DOX on 4T1 cells. (C) In vivo fluorescence images of mice before and
after intravenous injection of SP@DOX. (D) In vivo antimetastatic effects of SP@DOX in lung metastasis of breast cancer [18]. Copyright 2020, John Wiley and Sons.
(E) The schematic illustration of SP@AMF preparation and protection effects on intestine after applying cancer radiotherapy. In vivo protective effect of SP@AMF
against delayed (G) and early (F) injury caused by radiation with SP@AFM oral administration [16]. Copyright 2022, Nature Publishing Group.

Shchelik et al. [35] designed a two-step functionalization approach to
coat green algae, Chlamydomonas reinhardtii, to deliver antibiotic van-
comycin. The first step was coating with an NHS-dibenzocyclooctyne
anchor (DBCO) to functionalize the microalgae cell surface. Then, the
second step was loading vancomycin via biorthogonal click chemistry.
A photocleavable linker was used to control the antibiotic release, and
vancomycin could be free only upon UVA1. Another study on C. rein-
hardtii loading with vancomycin was reported by Studer et al. [36] re-
cently. Vancomycin was covalently connected to the functionalized sur-
face of C. reinhardtii by a disulfide-based linker and could be released
from the system with a thiol reductive agent. Moreover, Hosseini et al.
[37] engineered a multifunctional gold helix phototheranostic biohybrid
based on S. platensis for imaging-guided photothermal therapy (PTT).
Gold nanoparticles (AuNPs) were integrated with the S. platensis (Au-
SP) for computed tomography (CT) imaging, while L-cysteine-folic acid
(CF) was loaded to Au-SP (Au-SP@CF) to improve the theranostic effi-
cacy. Au-SP@CF could effectively inhibit the tumor for combating triple-
negative breast cancer and provides new insight into the theranostic sys-
tem for FL/CT-imaging-guided PTT (Fig. 3). In addition to improving
the drug delivery efficiency by artificial alteration, modifying microal-
gae surface with magnetic materials, such as magnetic nanoparticles,
to make it magnetotaxis has also become one of the research hotspots
[22,38-40]. We will minutely introduce microalgae drug delivery sys-
tems based on magnetotactic motion in a later section.

3
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 3. Surface modification-based microalgae drug delivery system. (A) The schematic illustration of targeting process and the synthesis of multifunctional gold
helix phototheranostic biohybrids and theranostic application of Au-SP@CF and dual-modal fluorescence and computed tomography-imaging-guided photothermal
therapy. (B) Computed tomography and fluorescence imaging of mice bearing T4 tumor. (C) In vivo imaging-guided photothermal therapeutic effects of Au-SP@CF
in alleviation triple-negative breast cancer [37]. Copyright 2022, American Chemical Society.

4
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
3. Applications of photosynthesis in microalgae as an oxygenator
Molecular oxygen is required for normal physiological metabolism
in most organism. Compared with normal blood vessels, tumor vessels
showed high permeability, even leakage [41]. Due to vascular leak-
age, some blood plasma is lost during the flow of blood from the ar-
teries to the veins, resulting in increased red blood cell concentration
and increased blood apparent viscosity. Therefore, the flow rate of red
blood cells in tumor blood vessels is an order of magnitude lower than
that in normal blood vessels [42]. These reasons are why hypoxic and
low-pH tumor microenvironment formation, which furtherly fuels ma-
lignant cell proliferation and accelerates tumor progression. Therefore,
numerous methods have been proposed to ameliorate tumor hypoxia
by improving oxygen concentration. For example, developing hypoxia-
activated prodrugs (HAPs) is one of the approaches, however, none of
HAPs are permitted by the FDA despite approximately 50 years of work
partly due to the overlapping toxicities of HAPs and traditional therapies
[43,44]. The test results of a combination of hyperbaric oxygen (HBO)
and RT are positive in cancer treatment [45]. However, some adverse
reactions may be combined with HBO treatment, such as oxygen toxi-
city, barotrauma, and decompression illness [46–48], and HBO has not
been a mainstream therapy so far. Recently, nanoparticles have been
used to produce oxygen in tumors to improve therapeutic efficacy, but
most of them could be trapped by the mononuclear phagocyte system,
which results in high doses and systemic toxicity limiting their appli-
cations in cancer treatment [49,50]. Microalgae can produce oxygen
through photosynthesis and are about 10 to 50 times more than land
plants [51]. Microalgae-medicated drug delivery systems not only have
the characteristics of other deliveries but also can produce oxygen in
situ. Recently, incremental attentions have been spending on the appli-
cations of microalgae in cancer treatment, and they have demonstrated
that microalgae can promote the therapeutic effect of tumor by tuning
its microenvironment.
3.1. Oxygen-dependent cancer therapy
Photodynamic therapy (PDT) and radiotherapy (RT) are two of the
vital cancer treatment, however, tumor hypoxia significantly limits the
efficacy of PDT and RT, which are oxygen-dependent cancer therapies
[52–57]. RT is a major treatment method in the early stages of can-
cer disease, using high-energy ionizing radiation for tumor elimination
[58,59]. However, tumor hypoxia significantly impairs RT efficacy, and
severe radiotherapy toxicity may be generated with a high X-ray dose
[60]. To promote the therapeutic efficacy of RT, Chai et al. [61] com-
bined Synechococcus elongates and two-dimensional (2D) bismuthene to
design a biomimetic radiosensitizer platform, which could effectively
improve the radiotherapy-resistant hypoxic tumor microenvironment.
In our previous research, C. vulgaris coated with engineered red blood
cell membrane (RBCM) is used to construct an oxygen-generating sys-
tem in cancer treatment (Fig. 4(A)). Oxygen was successfully generated
by RBCM-engineered algae in tumor tissues under the stimulation of
red light, which alleviates tumor hypoxia and increase sensitization to
RT [62]. Furthermore, PDT is currently becoming a well-known modal-
ity in the field of cancer treatment owning to its less invasiveness,
low toxicity, and lack of initiating resistance [54,63]. Shi’s team fab-
ricated two systems using cyanobacteria as a biotemplate to generate
oxygen and enhances type-II PDT efficacy. One study selected chlorin
e6 (ce6) as a photosensitizer to convert the oxygen to singlet oxygen
with 660 nm laser irradiation [64]. The other modified cyanobacteria
with inorganic two-dimensional black phosphorus nanosheets (BPNSs)
and perform the same function [65]. Zhang et al. [66] proposed using
upconversion nanoparticles (UCNPs) to improve microalgae photosyn-
thesis, further to enhance the therapeutic effect of PDT. The UCNPs were
bound with activated photosensitizer ce6, then conjugated with S. elon-
gatus PCC 7942 using a chemical modification. Additionally, Zhou et al.
[23] prepared an oxygen-affording material (ALGAE) using microalgae
5
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
Chlorella and calcium alginate, which can continuously and efficiently
supply oxygen around tumors. This system could achieve repetitive
PDT treatments, and effectively avoid the metastasis of triple-negative
breast cancer. Another research about Chlorella-based heterojunction-
engineered biohybrids was reported by Ou et al. [67] They modified
Chlorella with black phosphorus nanosheets (BPNSs) using polyaspar-
tic acid and Fe3+ mediated “Lego building method”. The photocatalysis
promoted the photosynthesis of Chlorella, thereby enhancing synergistic
photodynamic/chemodynamic/immune therapy. It is also demonstrated
by Wang et al. [68] that oxygen production enhanced the synergistic
therapeutic effect of the photodynamic/photothermally by microalgae-
based robots.
Additionally, it is common for microalgae to tune the tumor microen-
vironment in preclinical studies, which consequently enhances the effi-
cacy of RT/PDT during cancer treatment [22,24,69]. Our research team
used C. vulgaris to construct microalgae systems to improve tumor hy-
poxia by in-situ modulating hypoxia, thereby helping RT/PDT treat or-
thotropic breast cancer mice [20,60] (Fig. 4(B)). Furthermore, our an-
other research also introduced a biomineralized biohybrid microalgae
Algae@SiO2 relieving tumor hypoxia by light-mediated photosynthe-
sis, thereby improving the efficiency of RT/PDT therapy (Fig. 4(C)). C.
vulgaris was modified with silica using a simple one-step biomimetic
silicification method to construct Algae@SiO2 , which has demonstrated
excellent therapeutic effect in a mouse model bearing 4T1 breast tumor
[24]. Therefore, microalgae might be an excellent oxygenator during
oxygen-dependent tumor therapies.
However, due to the dependence of photosensitizer activation and
microalgae photosynthesis on visible light, these photo-regulated ther-
apies’ efficiency is severely limited in deep tissue [70]. In order to im-
prove the penetration of light, light-tunable materials are applied and
co-delivered with functional microalgae to promote the deposition of
light energy in the deep tissue [71]. UCNPs are ideal “partners” for med-
ical applications due to their intrinsic photon-converting characteristic,
which can transduce long-wavelength light into the visible light emis-
sion range [72,73]. The oxygen-producing capacity of microalgae can be
enhanced by UCNPs, and the photosynthesis microcapsules (PMCs) con-
structed by packing Synechocystis and UCNPs in alginate microcapsules
could constantly supply oxygen in tumor tissues [74] (Fig. 5). Huo et al.
[75] reported a near-infrared (NIR)-driven PDT platform. Photosensi-
tizer Rose Bengal (RB)-loaded UCNPs were hybridized with cyanobac-
teria Synechococcus elongatus PCC 7942. Red and green emissions up-
converted by the incident NIR laser could respectively enable the pho-
tosynthesis for oxygen evolution by the microalgal cells, and the pho-
tosensitization of the generated oxygen into singlet oxygen species for
effectively inhibiting tumors. This research provides a promising way
for largely enhanced PDT for deep-seated tumors by NIR upconversion-
mediated photosynthesis and photosensitization cascade. Therefore, co-
delivering UCNPs and microalgae is an efficient strategy to overcome
tumor hypoxia limitation in PDT against deep-seated tumors. Further-
more, Chang et al. [76] designed a novel CaAl2 O4 :Eu,Nd blue persis-
tent luminescence material (PLM) to continuously support the oxygen
production of cyanobacteria in vivo without the long-term external ex-
citation. Based on PLM and microalgae, they fabricated an exogenous
“irradiation-free” PDT platform to tune the tumor hypoxic microenvi-
ronment and promote the deep-seated tumor therapeutic effect. The
utilization of sustained light irradiation generated by persistent lumi-
nescence phosphors provides a promising strategy in oxygen-dependent
phototherapy with microalgae. Overall, oxygen-dependent cancer ther-
apies based on microalgae are still essential to be further explored to
contribute to the microalgae-based strategies in clinical practice.
3.2. Multimode synergistic therapy
Besides the application studies on oxygen-dependent cancer thera-
pies (RT and PDT), microalgae also have enormous potential in other
synergistic therapy, such as PTT, chemotherapy, immunotherapy, sono-
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 4. The application of photosynthesis of microalgae in cancer therapy. (A) The schematic illustration of Chlorella vulgaris (C. vulgaris) coated with red blood
cell membrane in alleviating tumor hypoxia under red light and producing ROS under laser irradiation. As well as the scanning electron microscope (SEM) photo
of C. vulgaris and C. vulgaris coated with red blood cell membrane [62]. Copyright 2020, American Association for the Advancement of Science. (B) The schematic
illustration of intravenously injected C. vulgaris@SiO2 in the 4T1 breast tumor-bearing mice and its generation of oxygen and ROS in breast tumors. The SEM pictures
are shown on the right [24]. Copyright 2020, American Chemical Society. (C) The mechanistic schematic illustration of intravenously injected C. vulgaris@CaP in
the synergistic therapy of cancer, and the schematic flow of the treatment of C. vulgaris@CaP in orthotopic tumor mice model [69]. Copyright 2021, Ivyspring
International Publisher.

dynamic therapy (SDT), and nanodynamic therapy (NDT). For instance,
endogenous pigments of microalgae could be used as photothermal
reagents to generate heat by converting light energy under the stimula-
tion of light irradiation [77]. Microalgae could generate heat under light
irradiation, thereby eliminating tumors and performing biophotother-
mal therapy (bio-PTT). Meanwhile, this smart PTT platform may also
release pathogen-associated adjuvants and molecular patterns for im-
mune stimulation. Wang et al. [78] developed a bio-PTT strategy based
on Synechococcus PCC 7942, which could inhibit tumors and reduce tu-
mor recurrence. However, the endogenous pigment accessibility as a
potent photothermal agent is impeded due to the limited photothermal
conversion efficiency. Therefore, gold nanomaterials have been used to
perform photoheat conversion [79]. For instance, Lee et al. [80] illus-
trate a hydrogel system containing Chlorella vulgaris and gold nanorods.
When the 4T1 tumor-bearing mice were intratumorally injected by this
system, abundant oxygen and oxygenated hemoglobin would be gen-
erated by this system stimulated by laser irradiation. Meanwhile, the
gold nanorods can increase the surrounding temperature and expand
blood vessels in tumors through responding to the near-infrared laser.
Furthermore, this study demonstrated that the combined therapy of this
system with DOX can efficiently improve breast cancer therapeutic ef-
fects (Fig. 6(A), (B)).
Tumor hypoxia seriously impacts the immune system and reduces
anti-tumor response due to the suppressed metabolic capacity of cyto-
toxic T lymphocyte (CTL) cells [81,82]. Tumor hypoxia results in the po-
larization of macrophages from M1 into M2 phenotype, thereby promot-
ing immunosuppressive cell proliferation and inducing the death of an-
titumor lymphocytes [83]. Thus, relieving tumor hypoxia is conducive
to enhancing synergistic photoimmunotherapy. For instance, cyanobac-
teria Synechococcus 7942 were used to fabricate a photosensitizer deliv-
ery system for tumor-targeted immunogenic PDT by Liu et al. [84] This
biomimetic system is able to alter the tumor immunosuppressive mi-
croenvironment, which enhance antitumor immune responses. Addi-
tionally, Wang et al. [85] also fabricated a Chlorella-based PDT-immune
synergistic platform modified with ce6 nanoparticles, which could en-
hance the boosts of PDT-induced immune responses and promote the
anti-tumor immune memory effect.
Besides, microalgae have been also applied in SDT and NDT. Very
recently, Gao et al. [86] developed a targeted drug delivery and sustain-
able oxygenator using surface-engineered Chlorella with the macrophage
membranes. Firstly, 𝛽-cyclodextrin (𝛽-CD) modified macrophage mem-
brane was used to coat onto Chlorella (CD-MChl). Then, adamantane-
modified liposome (ADA-NP) and CD-MChl were connected through
host-guest interactions to form supramolecular conjugates MChl-NP,
which could co-deliver hematoporphyrin (HP) and chloroquine phos-
phate (CQ) to the tumor tissues. Chlorella can continuously produce
oxygen to relieve the anoxic microenvironment of the tumor, and
the inflammatory-homing effects of macrophage membranes can help
this system increase biocompatibility and target tumor accumulation.
Therefore, this system can achieve the synergistic therapy of local

6
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
Fig. 5. The co-delivery of light-tunable material with microalgae in deep tissue treatment. (A) The schematic illustration of the structure of photosynthesis microcap-
sule (PMC). (B) Bioengineering and characterization of PMCs. (C) Therapeutic effects of NIR-PMCs combined with anti-PD-1 in breast cancer mice [74]. Copyright
2022, Nature Publishing Group.
oxygenation, sonodynamic therapy (SDT), and autophagy inhibition,
which could largely enhance the therapeutic efficacy against melanoma
(Fig. 6(C), (D)). Additionally, reactive oxygen species (ROS)-based NTD
has become an efficient approach in cancer therapy [87]. Lu et al.
[88] engineered a glutathione-depletion Mn(III)-riched manganese ox-
ide nanospikes (MnOx NSs) with cyanobacteria. This synergistic plat-
form could efficiently reform the tumor microenvironment via oxygen
production and GSH depletion. Interestingly, the cyanobacteria biohy-
brids were fabricated with bimetallic oxide Mn1.4 WOx nanosonosensi-
tizers, to conduct the photosynthetic oxygenation-augmented sonody-
namic nanotherapy of hypoxic tumors [89]. These microalgae-based
biohybrids provide new insight into photo-regulated multimode syner-
gistic therapies and offer the opportunity for clinical transformations.
4. Applications of locomotion in microalgae as microswimmer
4.1. Motility of microalgae
In addition to photosynthesis, some species of microalgae have the
ability to move. Because the nanotechnology, especially nanostructured
materials, has been rapidly developed, the research of microrobots based
on microalgae have gradually become one of the focuses in biomedical
applications. Microalgae, a biosafety microswimmer, provide new in-
sight and strategy for drug delivery [90]. Chlamydomonas reinhardtii,
a kind of diflagellate unicellular green algae, is one of the important
7
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
model organisms in microalgae research [91]. Zhang et al. [92,93] con-
structed two kinds of microrobots using the locomotion of C. reinhardtii
to deliver drugs to lung tissues and the gastrointestinal tract (GI), re-
spectively. Nanoparticle-modified microrobots were constructed to ac-
tively deliver antibiotics to the lungs [92]. The polymeric nanoparticles
loaded with ciprofloxacin (Cip) and coated with neutrophil membrane
were attached to C. reinhardtii using click chemistry. The microrobots
can move quickly (>110 μm/s) into the deep lung tissues, then they
can distribute uniformly and maintain for more than 2 days in it af-
ter intratracheal administration. In the acute Pseudomonas aeruginosa
pneumonia mouse model, the microrobots can significantly reduce the
mortality of pulmonary infection due to their outstanding characteris-
tics of long-time retention and continuous release of Cip. In the other
study, microalgae motors they constructed using C. reinhardtii were cov-
ered by a pH-sensitive capsule to protect them from the harsh gastric
environment. C. reinhardtii carrying DOX showed steady motion in in-
testinal fluid for more than 12 h after being released from the capsules,
thereby prolonging the DOX retention in the intestines [93]. In a recent
study, C. reinhardtii is also used by Studer et al. [36] to deliver the an-
tibiotic, vancomycin, which is attached to the surface of the microalgae
via a disulfide-based linker and released by thiol-based reducing agents.
However, the motility of the modified microalgae was only in vitro eval-
uated. These studies indicate that there are wide biomedical application
prospects to deliver drugs via microalgae with natural driving abilities,
however, these researches are just in their start-up stage.
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 6. The multimode synergistic effects of microalgae in cancer treatment. (A) The schematic illustration of the synthesis of Chlorella AuNRs BSA-Gel and its effect
on oxygen production and mild thermogenesis. (B) The thermogenesis ability of AuNRs BSA-Gel and the tumor volume of T4 tumor-bearing mice treated with AuNRs
BSA-Gel and Dox [80]. Copyright 2018, Elsevier. (C) The schematic illustration of MChl-CQ-HP-NP conjugate preparation, enhanced antitumor immunotherapy, and
tumor-targeted delivery. (D) In vivo enhanced sonodynamic therapy efficacy of MChl-CQ-HP-NP in the melanoma mouse model due to autophagy inhibition and
sustainable oxygenation [86]. Copyright 2023, American Chemical Society.

4.2. Phototaxis of microalgae
According to the different chemotacticum of microalgae, it can be
divided into phototaxis/photoavoidance, magnetotaxis, and chemotaxis
movement, thus, controllable movement of microalgae can be achieved
artificially. Weibel et al. [94] designed a “microoxen” to deliver micron-
sized polystyrene beads using microalgae, this microswimmer can be
guided to the target site with phototaxis/photoavoidance of C. rein-
hardtii, and the beads attached to the microalgae can be released by
UV light. In addition, Akolpoglu et al. [95] also used C. reinhardtii to
construct a microswimmer, which uses iron oxide nanoparticles coated
with chitosan and DOX to modify the surface of microalgae. It is proved
that the microswimmer still maintained the phototaxis and mobility of
the microalgae. Meanwhile, it also possesses the ability to conditionally
release the drug by using UV light. In vitro cell experiments proved that
the drug-loaded microswimmer could target and be effectively uptook
by SK-BR-3 tumor cells with light stimulation (Fig. 7).
4.3. Magnetotaxis of modified microalgae
In addition to the use of microalgae phototaxis/photoavoidance to
control their motion, many researchers have focused on their magne-
totaxis, i.e., using magnetic micro/nanoparticles to modify the surface
of microalgae which could furtherly help them to achieve directional
movement under the external magnetic field [22,38,40,68,96-99]. For
instance, Todd et al. [97] reported the first example of the in vivo tar-
geting the movement of diatoms through encapsulating with iron ox-
ide nanoparticles for magnetic delivery. Another research about diatom
magnetic modification used Fe3 O4 to fabricate biohybrid microrobots
via electrostatic adsorption for targeted drug delivery, and the move-
ment velocity of this system can go up to 28 μm/s with 3.5 mT magnetic
field strength. This system is also performed with high drug-loading ca-
pacity and pH-sensitive drug release [98]. Recently, Chlorella pyrenoi-
dosa participated in the designing of magnetic microrobot to deliver
drugs into targeted sites. Gong et al. [99] fabricated Chlorella-based
biohybrid microrobot multimers (BMMs) by using Fe3 O4 nanoparti-
cles deposited on the surface of microalgae cells. The self-assembled
BMMs showed excellent propulsion capability under magnetic actua-
tion, and achieved precise DOX delivery to enhance the chemother-
apy efficiency. Furthermore, C. reinhardtii also be used to construct a
biocompatible biohybrid microswimmer through surface noncovalent
binding with polyelectrolyte-functionalized magnetic spherical cargoes.
Active cargo delivery was confirmed by targeted transporting fluores-
cent isothiocyanate-dextran under the magnetic field [40]. A magnetic

8
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 7. The usage of the phototaxis of microal-

gae. (A) The schematic illustration of biohybrid
microswimmers (CSIONPs) preparation. (B) Char-
acterization of CSIONPs. (C) The visible light re-
sponse of CSIONPs. (D) In vitro drug delivery to
cancer cells by CSIONPs [95]. Copyright 2020,
John Wiley and Sons.

9
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
microrobot was designed by Wang et al. [38] using Spirulina called
Pd@Au)/Fe3O4@Sp.-DOX. Core-shell-structured Pd@Au nanoparticles
were inside Spirulina cells which used as photothermal conversion
agents, at the meantime, Fe3 O4 NPs nanoparticles were attached to the
surface of Spirulina to obtain magnetic actuating ability. Additionally,
Spirulina platensis was also used as the biotemplate by our group to de-
sign a photosynthetic biohybrid nanoswimmers system (PBNs) [22]. In
this system, superparamagnetic magnetite (Fe3 O4 NPs) was attached to
the surface of the microalgae and used to enable PBNs to target tumor
site and contribute to the magnetic resonance imaging. PBNs could di-
rectly move to anoxic 4T1 breast tumor tissues guided by an external
magnetic field, meanwhile, microalgae can generate oxygen in situ to
tune the tumor microenvironment, thus enhancing the effect of radio-
therapy (Fig. 8(A)). Another microalga, volvox, not only has phototaxis,
but also was fabricated as a magnetic microrobot (Volbot). Positively
charged magnetic nanoparticles (FeNp) were attached to the surface of
volvox by electrostatic absorption, thus Volbot can move regularly ac-
cording to pre-specified routes under the control of a magnetic field
[68] (Fig. 8(B)). Microalgae drug delivery systems are not limited to a
single function, but also have other functions such as locomotion, oxy-
gen production, and fluorescence imaging [22,68]. Thus, microalgae mi-
croswimmers are a great platform for cancer theranostic applications.
5. Applications of microalgae derivatives as drug carrier
Diatomite is a biogenic siliceous sedimentary rock formed by the de-
position of diatom microbial debris hundreds of millions of years ago.
Morse et al. [100] first proposed the idea of using diatomite as new
material with their genetically controlled precision of nanostructure.
The structure of diatomite achieved excellent therapeutic release prop-
erties in biomedicine and helped diatomite become an ideal material in
drug delivery. Jelena et al. [101] mixed diatomite with aluminum sul-
fate solution (Al2 (SO4 )3 ) to form inorganically functionalized diatomite,
then ibuprofen was loaded on it with a high drug loading efficiency
(201 mg/g), and a prolonged release time. Both physical and composite
mixtures of ibuprofen and modified diatomite performed better thera-
peutic effects at inhibiting pain in rats than the same dose of ibuprofen.
Also, chitosan/diatomite nanocomposite (CS/D) was constructed to en-
hance the diatomite properties of technical feasibility and biocompati-
bility as drug delivery [102]. Additionally, López-Cebral et al. [103] fab-
ricated chitosan/diatomite composite membranes enabling dual sublin-
gual drug delivery. Compared with 𝛽-chitosan membranes, this system
achieved higher drug load capacity, longer duration, and higher dose re-
lease. Besides oral administration, diatomite is also widely used in other
forms of drug delivery systems. Functionalized diatomite is an ideal ma-
terial to deliver drugs, which combines the advantages of the response
of diatomite properties to the drugs and the stimulation to the cell mi-
croenvironment. Functional materials commonly used to modify the sur-
face of diatoms include dopamine/Fe3 O4 [39], cyclic nitroxide 2,6,6-
tetramethylpiperidine-N-oxyl [104], Oligo (ethylene glycol) methacry-
lates [105], graphene oxide [106], chitosan [107], Id-peptide/siRNA
[108], polyethylene glycol/cell-penetrating peptide [109], vitamin B
[110], and so on. These materials were utilized to improve drug de-
livery efficiency, achieve sustained and controlled drug release, and
targeted drug delivery. For instance, the surface of diatoms were con-
nected with the dopamine-modified magnetic nanoparticles (Fe3 O4 ) by
one-step electrostatic self-assembly [39]. The magnetic properties of this
system were proved by using an external magnetic field to regulate its
motion. Then this system was used to deliver two drugs, indomethacin,
and gentamicin, to test its drug loading efficiency. To overcome the de-
naturation of antibodies in organic solvents, Delalat et al. [111] encap-
sulated hydrophobic drugs into liposomes and cationic micelles, then
attached them to the biosilica surface of engineered diatom Thalassiosira
pseudonana. In a subcutaneous xenograft neuroblastoma mouse model,
the application of the diatomite drug system led to tumour growth re-
gression, which indicated that diatomite could be used for targeted de-
10
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
livery of low water-soluble anticancer drugs to tumor sites. Overall, di-
atomite is a promising and stable drug delivery carrier. Moreover, the
three-dimensional porous structure of diatomite is considered to be an
extremely valuable natural template for micro/nanostructures of other
biomedical materials. Recently, the applications of diatomite in drug
delivery is increasing.
Many ingredients of microalgae, such as cell walls and membrane
vesicles, have also been utilized to fabricate drug delivery systems. For
example, Zorinc et al. [112] obtained reconstructed membrane vesicles
by the hypoosmotic shock of Dunaliella tertiolecta. These vesicles could
be used as sustainable drug carriers. Moreover, the microalgae Chlorella
sp. can also be used as a template to fabricate porous hollow hydroxyap-
atite microspheres (HHMs) via spray pyrolysis as a drug delivery system
[113]. Compared to non-HHMs, the highest ibuprofen loading capacity
was improved by 52 % with 0.893 g/g for HHMs, indicating that HHMs
are auspicious candidates to deliver drugs. Furthermore, García-Silva
et al. [114] synthesized microgels containing Schizochytrium sp. microal-
gae microgels and hybrid polyacrylic acid using the cell wall fraction as
a crosslinker, and bovine serum albumin was used to evaluate these mi-
crogels as antigen delivery carriers. Overall, microalgae are promising
biomaterials in drug delivery because of their especial structures and
bioactive substances. We firmly believe the drug delivery applications
of microalgae derivatives will continue to be searched.
6. Future perspectives
Despite important recent progress achieved over the past decade,
the drug delivery system based on natural microalgae is still in its start-
up stage. We consider that various gaps still need to be fulfilled before
these biomaterials can be clinically translated. Firstly, the biosafety of
microalgae needs further verification. Some studies have proved that
different administration methods of microalgae (oral and injection)
have good biocompatibility and biosafety. For instance, Zhang et al.
[16] proved that a month of daily gavage of Spirulina platensis did not
cause any adverse effects on main blood indexes and main organs in
mice. It was also shown that the main organs of mice did not have any
obvious inflammatory lesions or damage after 60 days of intravenous
injection with S. platensis. Moreover, the microalgae are proved to be
degraded and excreted out by the renal-urinary system as well [18,22].
However, the current research on microalgae biosafety is limited to
small animal models, such as mice. Therefore, more in vivo and in vitro
large animal models testing for the biosafety evaluation of microalgae
is needed before their clinical transformation. Secondly, the construc-
tion of bioengineered microalgae for antitumor immunotherapy may be
a promising development tendency. As discussed above, some strate-
gies have been taken to increase the tumor-targeting performance of
microalgae, such as using their phototaxis [94], being modified using
magnetic nanoparticles [22], or coating with macrophage membranes
[86]. However, there are few reports about bioengineered microalgae
in medicine, especially in targeted tumor therapy. The applications of
genetic engineering technology provide another possibility for microal-
gae transformation. Some researchers turned tumor-targeting bacteria
into drug-delivery vectors that can deliver proteins or nucleic acids to
the tumor site through genetic modifications [115]. These engineered
bacteria may be an excellent template for the development of bioengi-
neered microalgae construction for antitumor immunotherapy to de-
liver cytokines, chemokines, antigens, antibodies, and so on. Thirdly,
the studies about drug delivery systems based on microalgae should be
combined with the exploration of the function of their bioactive sub-
stances. In recent decades, the separation and purification technique of
bioactive substances in microalgae has been extensively studied [116].
The mechanisms of these bioactive substances of microalgae in certain
diseases have also been systematically explored [9]. However, when mi-
croalgae are delivered to the tissues as drug carriers, some intracellular
bioactive substances, such as extracellular vesicles, might be released
at the same time and play crucial functions. We consider that intensive
JID: ENGREG
ARTICLE IN PRESS [m5GeSdc;January 27, 2024;21:41]

H. Huang, Y. Lang, S. Wang et al. Engineered Regeneration xxx (xxxx) xxx

Fig. 8. The magnetotactic modification of microalgae. (A) The

schematic illustration of the modification of the magnetic S. platen-
sis in cancer therapy, and the morphology of S. platensis in scan-
ning electron microscopy (SEM) images (left) and fluorescent im-
ages (right) [22] Copyright 2020, John Wiley and Sons. (B) Char-
acterization of the Volbot and the schematic illustration of image-
guided photodynamic/photothermal combination cancer therapy
based on Volbot. (C) The schematic illustration of the Volbot fabri-
cated by electrostatic absorption and characterization of the Volbot
guided by the external magnetic field [68]. Copyright 2022, John
Wiley and Sons.

11
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
studies on the biological function of microalgae may promote their de-
velopment in the domain of drug delivery.
7. Conclusions
Owning to their special morphological characteristics and easily
modified surfaces, microalgae are becoming a new hotspot in medicine,
and hold excellent potential as a novel drug delivery carrier. Microalgae
possess unique characteristics as a drug delivery system, these include
photosynthesis, active surface, high biocompatibility, biodegradability,
and micron size of microalgae. This review article mainly presents the
applications of microalgae, especially how they are utilized to perform
the process of drug delivery. Numerous evidences have been proved mi-
croalgae is an ideal drug vehicle, and these researches encourage the
possibility of grand evolutions in the field of drug delivery. We believe
that significant advances in microalgae-based drug delivery have been
made, but we are still far from where we should be, and this is still in
its infancy.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Hui Huang: Conceptualization, Investigation, Writing – original
draft. Yutong Lang: Investigation, Writing – original draft. Shoujie
Wang: Writing – review & editing. Min Zhou: Conceptualization, Writ-
ing – review & editing, Supervision, Resources, Funding acquisition.
Acknowledgment
This work was supported by the National Key R&D Program of China
(2022YFA1104900), the Leading Innovative and Entrepreneur Team In-
troduction Program of Zhejiang (2022R01002), the Binjiang Institute
of Zhejiang University (ZY202205SMKY007), and the Natural Science
Foundation of Shandong Province (ZR2023ZD30).
References
[1] Y.H. Yun, B.K. Lee, K. Park, Controlled drug delivery: historical perspective for the
next generation, J. Control. Release 219 (2015) 2–7.
[2] R. Langer, Drug delivery and targeting, Nature 392 (1998) 5–10.
[3] A.M. Vargason, A.C. Anselmo, S. Mitragotri, The evolution of commercial drug
delivery technologies, Nat. Biomed. Eng. 5 (2021) 951–967.
[4] O.O. Ige, L.E. Umoru, S. Aribo, Natural products: a minefield of biomaterials, ISRN
Mater. Sci. 2012 (2012) 1–20.
[5] P.C. Pires, F. Mascarenhas-Melo, K. Pedrosa, D. Lopes, J. Lopes, A. Macário-Soares,
D. Peixoto, P.S. Giram, F. Veiga, A.C. Paiva-Santos, Polymer-based biomaterials for
pharmaceutical and biomedical applications: a focus on topical drug administra-
tion, Eur. Polym. J. 187 (2023) 111868.
[6] A. Sood, A. Gupta, G. Agrawal, Recent advances in polysaccharides based bioma-
terials for drug delivery and tissue engineering applications, Carbohydr. Polym.
Technol. Appl. 2 (2021) 100067.
[7] E. Shalaby, Algae as promising organisms for environment and health, Plant Signal.
Behav. 6 (2011) 1338–1350.
[8] Y. Sumi, in: Microalgae Pioneering the Future-Application And Utilization, NISTEP
Science & Technology Foresight Center, 2009, pp. 1349–3663.
[9] D. Zhuang, N. He, K.S. Khoo, E.P. Ng, K.W. Chew, T.C. Ling, Application progress
of bioactive compounds in microalgae on pharmaceutical and cosmetics, Chemo-
sphere 291 (2022) 132932.
[10] S. Basheer, S. Huo, F. Zhu, J. Qian, L. Xu, F. Cui, B. Zou, in: Microalgae in Human
Health and Medicine, Microalgae Biotechnology for Food, Health and High Value
Products, Springer Nature Singapore Pte Ltd, 2020, pp. 149–174.
[11] R. Sathasivam, R. Radhakrishnan, A. Hashem, E.F. Abd_Allah, Microalgae metabo-
lites: a rich source for food and medicine, Saudi J. Biol. Sci. 26 (2019) 709–722.
[12] D. Zhong, Z. Du, M. Zhou, Algae: a natural active material for biomedical applica-
tions, View 2 (2021) 20200189.
[13] S. Liu, H. Yang, M.Y. Ho, B. Xing, Recent advances of material-decorated photo-
synthetic microorganisms and their aspects in biomedical applications, Adv. Opt.
Mater. 11 (2023) 2203038.
12
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
[14] U.T. Uthappa, V. Brahmkhatri, G. Sriram, H.-Y. Jung, J. Yu, N. Kurkuri, T.M. Am-
inabhavi, T. Altalhi, G.M. Neelgund, M.D. Kurkuri, Nature engineered diatom
biosilica as drug delivery systems, J. Control. Release 281 (2018) 70–83.
[15] J. Delasoie, F. Zobi, Natural diatom biosilica as microshuttles in drug delivery sys-
tems, Pharmaceutics 11 (2019) 537.
[16] D. Zhang, D. Zhong, J. Ouyang, J. He, Y. Qi, W. Chen, X. Zhang, W. Tao, M. Zhou,
Microalgae-based oral microcarriers for gut microbiota homeostasis and intestinal
protection in cancer radiotherapy, Nat. Commun. 13 (2022) 1413.
[17] D. Zhong, D. Zhang, W. Chen, J. He, C. Ren, X. Zhang, N. Kong, W. Tao, M. Zhou,
Orally deliverable strategy based on microalgal biomass for intestinal disease treat-
ment, Sci. Adv. 7 (2021) eabi9265.
[18] D. Zhong, D. Zhang, T. Xie, M. Zhou, Biodegradable microalgae-based carriers for
targeted delivery and imaging-guided therapy toward lung metastasis of breast
cancer, Small 16 (2020) 2000819.
[19] S. Xie, N. Jiao, S. Tung, L. Liu, Controlled regular locomotion of algae cell micro-
robots, Biomed. Microdevices 18 (2016) 1–9.
[20] W. Li, S. Wang, D. Zhong, Z. Du, M. Zhou, A bioactive living hydrogel: photo-
synthetic bacteria mediated hypoxia elimination and bacteria-killing to promote
infected wound healing, Adv. Therap. 4 (2021) 2000107.
[21] H. Hu, D. Zhong, W. Li, X. Lin, J. He, Y. Sun, Y. Wu, M. Shi, X. Chen, F. Xu, M. Zhou,
Microalgae-based bioactive hydrogel loaded with quorum sensing inhibitor pro-
motes infected wound healing, Nano Today 42 (2022) 101368.
[22] D. Zhong, W. Li, Y. Qi, J. He, M. Zhou, Photosynthetic biohybrid nanoswimmers
system to alleviate tumor hypoxia for FL/PA/MR imaging-guided enhanced ra-
dio-photodynamic synergetic therapy, Adv. Funct. Mater. 30 (2020) 1910395.
[23] T.-J. Zhou, L. Xing, Y.-T. Fan, P.-F. Cui, H.-L. Jiang, Light triggered oxygen-af-
fording engines for repeated hypoxia-resistant photodynamic therapy, J. Control.
Release 307 (2019) 44–54.
[24] W. Li, D. Zhong, S. Hua, Z. Du, M. Zhou, Biomineralized biohybrid algae for tumor
hypoxia modulation and cascade radio-photodynamic therapy, ACS Appl. Mater.
Interfaces 12 (2020) 44541–44553.
[25] D. Vona, M.L. Presti, S.R. Cicco, F. Palumbo, R. Ragni, G.M. Farinola, Light emit-
ting silica nanostructures by surface functionalization of diatom algae shells with
a triethoxysilane-functionalized 𝜋-conjugated fluorophore, MRS Adv. 1 (2016)
3817–3823.
[26] J. Chen, G. Qin, Q. Chen, J. Yu, S. Li, F. Cao, B. Yang, Y. Ren, A synergistic combi-
nation of diatomaceous earth with Au nanoparticles as a periodically ordered, but-
ton-like substrate for SERS analysis of the chemical composition of eccrine sweat
in latent fingerprints, J. Mater. Chem. C 3 (2015) 4933–4944.
[27] S. Managò, G. Zito, A. Rogato, M. Casalino, E. Esposito, A.C. De Luca, E. De Tom-
masi, Bioderived three-dimensional hierarchical nanostructures as efficient sur-
face-enhanced Raman scattering substrates for cell membrane probing, ACS Appl.
Mater. Interfaces 10 (2018) 12406–12416.
[28] U. Hopfner, T.-L. Schenck, M.-N. Chávez, H.-G. Machens, A.-V. Bohne, J. Nickelsen,
R.-E. Giunta, J.-T. Egaña, Development of photosynthetic biomaterials for in vitro
tissue engineering, Acta Biomater. 10 (2014) 2712–2717.
[29] Y. Haraguchi, Y. Kagawa, K. Sakaguchi, K. Matsuura, T. Shimizu, T. Okano, Thicker
three-dimensional tissue from a “symbiotic recycling system” combining mam-
malian cells and algae, Sci. Rep. 7 (2017) 1–10.
[30] D. Steffens, M. Lersch, A. Rosa, C. Scher, T. Crestani, M. Morais, J. Costa, P. Pranke,
A new biomaterial of nanofibers with the microalga Spirulinaas scaffolds to cultivate
with stem cells for use in tissue engineering, J. Biomed. Nanotechnol. 9 (2013)
710–718.
[31] M. Bariana, M.S. Aw, M. Kurkuri, D. Losic, Tuning drug loading and release prop-
erties of diatom silica microparticles by surface modifications, Int. J. Pharm. 443
(2013) 230–241.
[32] C. Mohanty, M. Das, S.K. Sahoo, Emerging role of nanocarriers to increase the
solubility and bioavailability of curcumin, Expert Opin. Drug Deliv. 9 (2012)
1347–1364.
[33] S. Banerjee, J. Pillai, Solid lipid matrix mediated nanoarchitectonics for improved
oral bioavailability of drugs, Expert Opin. Drug Metab. Toxicol. 15 (2019) 499–515.
[34] P. Gnanamoorthy, S. Anandhan, V.A. Prabu, Natural nanoporous silica frustules
from marine diatom as a biocarrier for drug delivery, J. Porous Mater. 21 (2014)
789–796.
[35] I.S. Shchelik, S. Sieber, K. Gademann, Green algae as a drug delivery system for
the controlled release of antibiotics, Chem. Eur. J. 26 (2020) 16644–16648.
[36] T. Studer, D. Morina, I.S. Shchelik, K. Gademann, Biohybrid microswimmers for
antibiotic drug delivery based on a thiol-sensitive release platform, Chem. Eur. J.
29 (2023) e202203913.
[37] M. Hosseini, Z. Ahmadi, A. Kefayat, F. Molaabasi, A. Ebrahimpour, Y. Naderi Kho-
jasteh, M. Khoobi, Multifunctional gold helix phototheranostic biohybrid that en-
ables targeted image-guided photothermal therapy in breast cancer, ACS Appl.
Mater. Interfaces 14 (2022) 37447–37465.
[38] X. Wang, J. Cai, L. Sun, S. Zhang, D. Gong, S. Yue, L. Feng, D. Zhang, Facile fab-
rication of magnetic microrobots based on spirulina templates for targeted deliv-
ery and synergistic chemo-photothermal therapy, ACS Appl. Mater. Interfaces 11
(2019) 4745–4756.
[39] D. Losic, Y. Yu, M.S. Aw, S. Simovic, B. Thierry, J. Addai-Mensah, Surface func-
tionalisation of diatoms with dopamine modified iron-oxide nanoparticles: toward
magnetically guided drug microcarriers with biologically derived morphologies,
Chem. Commun. 46 (2010) 6323–6325.
[40] O. Yasa, P. Erkoc, Y. Alapan, M. Sitti, Microalga-powered microswimmers toward
active cargo delivery, Adv. Mater. 30 (2018) 1804130.
[41] M. Ramazanilar, A. Mojra, Characterization of breast tissue permeability for de-
tection of vascular breast tumors: an in vitro study, Mater. Sci. Eng. C 107 (2020)
110222.
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
[42] E.P. Troendle, A. Khan, P.C. Searson, M.B. Ulmschneider, Predicting drug delivery
efficiency into tumor tissues through molecular simulation of transport in complex
vascular networks, Biophys. J. 114 (2018) 679a.
[43] J.J. Yeh, W.Y. Kim, Targeting tumor hypoxia with hypoxia-activated prodrugs, J.
Clin. Oncol. 33 (2015) 1505–1508.
[44] I.N. Mistry, M. Thomas, E.D. Calder, S.J. Conway, E.M. Hammond, Clinical ad-
vances of hypoxia-activated prodrugs in combination with radiation therapy, Int.
J. Radiat. Oncol. Biol. Phys. 98 (2017) 1183–1196.
[45] K. Stępień, R.P. Ostrowski, E. Matyja, Hyperbaric oxygen as an adjunctive therapy
in treatment of malignancies, including brain tumours, Med. Oncol. 33 (2016) 1–9.
[46] M. Heyboer III, D. Sharma, W. Santiago, N. McCulloch, Hyperbaric oxygen therapy:
side effects defined and quantified, Adv. Wound Care 6 (2017) 210–224.
[47] A.E. Gunes, O. Gozeneli, A. Akal, M.E. Guldur, E. Savik, Reduction of side effects of
hyperbaric oxygen therapy with thymoquinone treatment in rats, Undersea Hyperb.
Med. 44 (2017) 337–343.
[48] E.M. Camporesi, Side effects of hyperbaric oxygen therapy, Undersea Hyperb. Med.
41 (2014) 253–257.
[49] S. Singh, A. Sharma, G.P. Robertson, Realizing the clinical potential of cancer nan-
otechnology by minimizing toxicologic and targeted delivery concerns limitations
and approaches of cancer nanotechnology, Cancer Res. 72 (2012) 5663–5668.
[50] S. Sharma, R. Parveen, B.P. Chatterji, Toxicology of nanoparticles in drug delivery,
Curr. Pathobiol. Rep. 9 (2021) 1–12.
[51] W. Zhou, J. Wang, P. Chen, C. Ji, Q. Kang, B. Lu, K. Li, J. Liu, R. Ruan, Bio-mitiga-
tion of carbon dioxide using microalgal systems: advances and perspectives, Renew.
Sustain. Energy Rev. 76 (2017) 1163–1175.
[52] B. Ji, M. Wei, B. Yang, Recent advances in nanomedicines for photodynamic ther-
apy (PDT)-driven cancer immunotherapy, Theranostics 12 (2022) 434.
[53] M. Busk, J. Overgaard, M.R. Horsman, Imaging of tumor hypoxia for radiotherapy:
current status and future directions, Semin. Nucl. Med. 50 (2020) 562–583.
[54] H. Cui, Y. Su, W. Wei, F. Xu, J. Gao, W. Zhang, How microalgae is effective in
oxygen deficiency aggravated diseases? A comprehensive review of literature, Int.
J. Nanomed. 17 (2022) 3101–3122.
[55] K. Graham, E. Unger, Overcoming tumor hypoxia as a barrier to radiotherapy,
chemotherapy and immunotherapy in cancer treatment, Int. J. Nanomed. 13 (2018)
6049.
[56] M.V. Blagosklonny, Antiangiogenic therapy and tumor progression, Cancer Cell 5
(2004) 13–17.
[57] H.E. Barker, J.T. Paget, A.A. Khan, K.J. Harrington, The tumour microenvironment
after radiotherapy: mechanisms of resistance and recurrence, Nat. Rev. Cancer 15
(2015) 409–425.
[58] J.-F. Bosset, L. Collette, G. Calais, L. Mineur, P. Maingon, L. Radosevic-Jelic, A. Da-
ban, E. Bardet, A. Beny, J.-C. Ollier, Chemotherapy with preoperative radiotherapy
in rectal cancer, N. Engl. J. Med. 355 (2006) 1114–1123.
[59] X. Chen, J. Song, X. Chen, H. Yang, X-ray-activated nanosystems for theranostic
applications, Chem. Soc. Rev. 48 (2019) 3073–3101.
[60] D. De Ruysscher, G. Niedermann, N.G. Burnet, S. Siva, A.W. Lee, F. Hegi-Johnson,
Radiotherapy toxicity, Nat. Rev. Dis. Primers 5 (2019) 13.
[61] R. Chai, L. Yu, C. Dong, Y. Yin, S. Wang, Y. Chen, Q. Zhang, Oxygen-evolving
photosynthetic cyanobacteria for 2D bismuthene radiosensitizer-enhanced cancer
radiotherapy, Bioact. Mater. 17 (2022) 276–288.
[62] Y. Qiao, F. Yang, T. Xie, Z. Du, D. Zhong, Y. Qi, Y. Li, W. Li, Z. Lu, J. Rao, Y. Sun,
M. Zhou, Engineered algae: a novel oxygen-generating system for effective treat-
ment of hypoxic cancer, Sci. Adv. 6 (2020) eaba5996.
[63] S. Yano, S. Hirohara, M. Obata, Y. Hagiya, S.-i. Ogura, A. Ikeda, H. Kataoka,
M. Tanaka, T. Joh, Current states and future views in photodynamic therapy, J.
Photochem. Photobiol. C 12 (2011) 46–67.
[64] M. Huo, L. Wang, L. Zhang, C. Wei, Y. Chen, J. Shi, Photosynthetic tumor oxy-
genation by photosensitizer-containing cyanobacteria for enhanced photodynamic
therapy, Angew. Chem. Int. Ed. 59 (2020) 1906–1913.
[65] F. Qi, P. Ji, Z. Chen, L. Wang, H. Yao, M. Huo, J. Shi, Photosynthetic cyanobac-
teria-hybridized black phosphorus nanosheets for enhanced tumor photodynamic
therapy, Small 17 (2021) 2102113.
[66] Y. Zhang, H. Liu, X. Dai, H. Li, X. Zhou, S. Chen, J. Zhang, X.-J. Liang, Z. Li,
Cyanobacteria-based near-infrared light-excited self-supplying oxygen system for
enhanced photodynamic therapy of hypoxic tumors, Nano Res. 14 (2021) 667–673.
[67] M. Ou, C. Lin, Y. Wang, Y. Lu, W. Wang, Z. Li, W. Zeng, X. Zeng, X. Ji, L. Mei, Het-
erojunction engineered bioactive chlorella for cascade promoted cancer therapy,
J. Control. Release 345 (2022) 755–769.
[68] J. Wang, F. Soto, S. Liu, Q. Yin, E. Purcell, Y. Zeng, E.-C. Hsu, D. Akin, B. Sinclair,
T. Stoyanova, U. Demirci, Volbots: volvox microalgae-based robots for multimode
precision imaging and therapy, Adv. Funct. Mater. 32 (2022) 2201800.
[69] D. Zhong, W. Li, S. Hua, Y. Qi, T. Xie, Y. Qiao, M. Zhou, Calcium phosphate engi-
neered photosynthetic microalgae to combat hypoxic-tumor by in-situ modulating
hypoxia and cascade radio-phototherapy, Theranostics 11 (2021) 3580.
[70] Z. Zhang, Q. Han, J.W. Lau, B. Xing, Lanthanide-doped upconversion nanoparticles
meet the needs for cutting-edge bioapplications: recent progress and perspectives,
ACS Mater. Lett. 2 (2020) 1516–1531.
[71] Q. Han, J.W. Lau, T.C. Do, Z. Zhang, B. Xing, Near-infrared light brightens bacte-
rial disinfection: recent progress and perspectives, ACS Appl. Bio Mater. 4 (2020)
3937–3961.
[72] H. Chen, B. Ding, J. Lin, Recent progress in upconversion nanomaterials for emerg-
ing optical biological applications, Adv. Drug Deliv. Rev. 188 (2022) 114414.
[73] X. Ai, C.J.H. Ho, J. Aw, A.B.E. Attia, J. Mu, Y. Wang, X. Wang, Y. Wang, X. Liu,
H. Chen, M. Gao, X. Chen, E.K.L. Yeow, G. Liu, M. Olivo, B. Xing, In vivo covalent
cross-linking of photon-converted rare-earth nanostructures for tumour localization
and theranostics, Nat. Commun. 7 (2016) 10432.
13
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
[74] W. Wang, H. Zheng, J. Jiang, Z. Li, D. Jiang, X. Shi, H. Wang, J. Jiang, Q. Xie,
M. Gao, J. Chu, X. Cai, T. Xia, R. Li, Engineering micro oxygen factories to slow
tumour progression via hyperoxic microenvironments, Nat. Commun. 13 (2022)
4495.
[75] M. Huo, P. Liu, L. Zhang, C. Wei, L. Wang, Y. Chen, J. Shi, Upconversion nanoparti-
cles hybridized cyanobacterial cells for near-infrared mediated photosynthesis and
enhanced photodynamic therapy, Adv. Funct. Mater. 31 (2021) 2010196.
[76] M. Chang, W. Feng, L. Ding, H. Zhang, C. Dong, Y. Chen, J. Shi, Persistent lumines-
cence phosphor as in-vivo light source for tumoral cyanobacterial photosynthetic
oxygenation and photodynamic therapy, Bioact. Mater. 10 (2022) 131–144.
[77] R. Goss, B. Lepetit, Biodiversity of NPQ, J. Plant Physiol. 172 (2015) 13–32.
[78] H. Wang, Y. Guo, S. Gan, H. Liu, Q. Chen, A. Yuan, Y. Hu, J. Wu, Photosynthetic
microorganisms-based biophotothermal therapy with enhanced immune response,
Small 17 (2021) 2007734.
[79] J.B. Vines, J.-H. Yoon, N.-E. Ryu, D.-J. Lim, H. Park, Gold nanoparticles for pho-
tothermal cancer therapy, Front. Chem. 7 (2019) 167.
[80] C. Lee, K. Lim, S.S. Kim, E.S. Lee, K.T. Oh, H.-G. Choi, Y.S. Youn, Chlorella-gold
nanorods hydrogels generating photosynthesis-derived oxygen and mild heat for
the treatment of hypoxic breast cancer, J. Control. Release 294 (2019) 77–90.
[81] A.E. Nejad, S. Najafgholian, A. Rostami, A. Sistani, S. Shojaeifar, M. Esparvarinha,
R. Nedaeinia, S.H. Javanmard, M. Taherian, M. Ahmadlou, R. Salehi, B. Sadeghi,
M. Manian, The role of hypoxia in the tumor microenvironment and development
of cancer stem cell: a novel approach to developing treatment, Cancer Cell Int. 21
(2021) 1–26.
[82] P. Yotnda, D. Wu, A.M. Swanson, Hypoxic tumors and their effect on immune cells
and cancer therapy, Methods Mol. Biol. 651 (2010) 1–29.
[83] S. Yan, G. Wan, Tumor-associated macrophages in immunotherapy, FEBS J. 288
(2021) 6174–6186.
[84] L. Liu, H. He, Z. Luo, H. Zhou, R. Liang, H. Pan, Y. Ma, L. Cai, In Situ photocat-
alyzed oxygen generation with photosynthetic bacteria to enable robust immuno-
genic photodynamic therapy in triple-negative breast cancer, Adv. Funct. Mater.
30 (2020) 1910176.
[85] H. Wang, H. Liu, Y. Guo, W. Zai, X. Li, W. Xiong, X. Zhao, Y. Yao, Y. Hu, Z. Zou,
J. Wu, et al., Photosynthetic microorganisms coupled photodynamic therapy for
enhanced antitumor immune effect, Bioact. Mater. 12 (2022) 97–106.
[86] C. Gao, C.H. Kwong, Q. Wang, H. Kam, B. Xie, S.M.-Y. Lee, G. Chen, R. Wang,
Conjugation of macrophage-mimetic microalgae and liposome for antitumor son-
odynamic immunotherapy via hypoxia alleviation and autophagy inhibition, ACS
Nano 17 (2023) 4034–4049.
[87] Q. Xu, Y. Yang, J. Lu, Y. Lin, S. Feng, X. Luo, D. Di, S. Wang, Q. Zhao, Recent
trends of mesoporous silica-based nanoplatforms for nanodynamic therapies, Co-
ord. Chem. Rev. 469 (2022) 214687.
[88] S. Lu, W. Feng, X. Yao, X. Song, J. Guo, Y. Chen, Z. Hu, Microorganism-enabled
photosynthetic oxygeneration and ferroptosis induction reshape tumor microenvi-
ronment for augmented nanodynamic therapy, Biomaterials 287 (2022) 121688.
[89] S. Lu, W. Feng, C. Dong, X. Song, X. Gao, J. Guo, Y. Chen, Z. Hu, Photosynthetic oxy-
genation-augmented sonodynamic nanotherapy of hypoxic tumors, Adv. Healthc.
Mater. 11 (2022) 2102135.
[90] H. Xin, N. Zhao, Y. Wang, X. Zhao, T. Pan, Y. Shi, B. Li, Optically controlled living
micromotors for the manipulation and disruption of biological targets, Nano Lett.
20 (2020) 7177–7185.
[91] N.T. Tran, R. Kaldenhoff, Achievements and challenges of genetic engineering of
the model green alga Chlamydomonas reinhardtii, Algal Res. 50 (2020) 101986.
[92] F. Zhang, J. Zhuang, Z. Li, H. Gong, B.E.-F. de Ávila, Y. Duan, Q. Zhang, J. Zhou,
L. Yin, E. Karshalev, W. Gao, V. Nizet, R.H. Fang, L. Zhang, J. Wang, Nanopar-
ticle-modified microrobots for in vivo antibiotic delivery to treat acute bacterial
pneumonia, Nat. Mater. 21 (2022) 1324–1332.
[93] F. Zhang, Z. Li, Y. Duan, A. Abbas, R. Mundaca-Uribe, L. Yin, H. Luan, W. Gao,
R.H. Fang, J. Wand, Gastrointestinal tract drug delivery using algae motors em-
bedded in a degradable capsule, Sci. Robot. 7 (2022) eabo4160.
[94] D.B. Weibel, P. Garstecki, D. Ryan, W.R. DiLuzio, M. Mayer, J.E. Seto, G.M. White-
sides, Microoxen: microorganisms to move microscale loads, Proc. Natl. Acad. Sci.
102 (2005) 11963–11967.
[95] M.B. Akolpoglu, N.O. Dogan, U. Bozuyuk, H. Ceylan, S. Kizilel, M. Sitti, High-
-yield production of biohybrid microalgae for on-demand cargo delivery, Adv. Sci.
7 (2020) 2001256.
[96] D. Gong, N. Celi, L. Xu, D. Zhang, J. Cai, CuS nanodots-loaded biohybrid mag-
netic helical microrobots with enhanced photothermal performance, Mater. Today
Chem. 23 (2022) 100694.
[97] T. Todd, Z. Zhen, W. Tang, H. Chen, G. Wang, Y.-J. Chuang, K. Deaton, Z. Panc,
J. Xie, Iron oxide nanoparticle encapsulated diatoms for magnetic delivery of small
molecules to tumors, Nanoscale 6 (2014) 2073–2076.
[98] M. Li, J. Wu, D. Lin, J. Yang, N. Jiao, Y. Wang, L. Liu, A diatom-based biohybrid
microrobot with a high drug-loading capacity and pH-sensitive drug release for
target therapy, Acta Biomater. 154 (2022) 443–453.
[99] D. Gong, N. Celi, D. Zhang, J. Cai, Magnetic biohybrid microrobot multimers based
on chlorella cells for enhanced targeted drug delivery, ACS Appl. Mater. Interfaces
14 (2022) 6320–6330.
[100] S. Maher, T. Kumeria, M.S. Aw, D. Losic, Diatom silica for biomedical applications:
recent progress and advances, Adv. Healthc. Mater. 7 (2018) 1800552.
[101] J. Janićijević, J. Milić, B. Čalija, A. Micov, R. Stepanović-Petrović, M. Tomić,
A. Daković, V. Dobričić, B. Vasiljeviće, D. Krajišnika, Potentiation of the ibupro-
fen antihyperalgesic effect using inorganically functionalized diatomite, J. Mater.
Chem. B 6 (2018) 5812–5822.
[102] S.M. Ibrahim, M.N. Bin Jumah, S.I. Othman, R.S. Alruhaimi, N. Al-Khalawi,
Y.F. Salama, A.A. Allam, M.R. Abukhadra, Synthesis of chitosan/diatomite com-
JID: ENGREG
ARTICLE IN PRESS
H. Huang, Y. Lang, S. Wang et al.
posite as an advanced delivery system for ibuprofen drug; equilibrium studies and
the release profile, ACS Omega 6 (2021) 13406–13416.
[103] R. López-Cebral, G. Peng, L.L. Reys, S.S. Silva, J.M. Oliveira, J. Chen, T.H. Silva,
R.L. Reiset, Dual delivery of hydrophilic and hydrophobic drugs from chi-
tosan/diatomaceous earth composite membranes, J. Mater. Sci. Mater. Med. 29
(2018) 1–12.
[104] S.R. Cicco, D. Vona, E. De Giglio, S. Cometa, M. Mattioli-Belmonte, F. Palumbo,
R. Ragni, G.M. Farinola, Chemically modified diatoms biosilica for bone cell growth
with combined drug-delivery and antioxidant properties, Chempluschem 80 (2015)
1104–1112.
[105] R. Vasani, D. Losic, A. Cavallaro, N. Voelcker, Fabrication of stimulus-responsive
diatom biosilica microcapsules for antibiotic drug delivery, J. Mater. Chem. B 3
(2015) 4325–4329.
[106] T. Kumeria, M. Bariana, T. Altalhi, M. Kurkuri, C.T. Gibson, W. Yang, D. Losic,
Graphene oxide decorated diatom silica particles as new nano-hybrids: towards
smart natural drug microcarriers, J. Mater. Chem. B 1 (2013) 6302–6311.
[107] R. Sasirekha, T.S. Sheena, M.S. Deepika, P. Santhanam, H.E. Townley, K. Je-
ganathan, S.D. Kumar, K. Premkumar, Surface engineered Amphora subtropica
frustules using chitosan as a drug delivery platform for anticancer therapy, Mater.
Sci. Eng. C Mater. Biol. Appl. 94 (2019) 56–64.
[108] N.M. Martucci, N. Migliaccio, I. Ruggiero, F. Albano, G. Calì, S. Romano, M. Terrac-
ciano, I. Rea, P. Arcari, A. Lamberti, Nanoparticle-based strategy for personalized
B-cell lymphoma therapy, Int. J. Nanomed. 11 (2016) 6089.
[109] M. Terracciano, M.-A. Shahbazi, A. Correia, I. Rea, A. Lamberti, L. De Stefano,
H.A. Santos, Surface bioengineering of diatomite based nanovectors for efficient
intracellular uptake and drug delivery, Nanoscale 7 (2015) 20063–20074.
14
[m5GeSdc;January 27, 2024;21:41]
Engineered Regeneration xxx (xxxx) xxx
[110] J. Delasoie, J. Rossier, L. Haeni, B. Rothen-Rutishauser, F. Zobi, Slow-targeted re-
lease of a ruthenium anticancer agent from vitamin B12 functionalized marine
diatom microalgae, Dalton Trans. 47 (2018) 17221–17232.
[111] B. Delalat, V.C. Sheppard, S.R. Ghaemi, S. Rao, C.A. Prestidge, G. McPhee,
M.-L. Rogers, J.F. Donoghue, V. Pillay, T.G. Johns, N. Kröger, N.H. Voelcker, Tar-
geted drug delivery using genetically engineered diatom biosilica, Nat. Commun.
6 (2015) 8791.
[112] M.L. Zorinc, I. Demir-Yilmaz, C. Formosa-Dague, I. Vrana, B. Gašparović, L. Horvat,
A. Butorac, R. Frkanec, N.I. DeNardis, Reconstructed membrane vesicles from the
microalga Dunaliella as a potential drug delivery system, Bioelectrochemistry 150
(2023) 108360.
[113] N.S. Sambudi, S. Cho, K. Cho, Porous hollow hydroxyapatite microspheres synthe-
sized by spray pyrolysis using a microalga template: preparation, drug delivery,
and bioactivity, RSC Adv. 6 (2016) 43041–43048.
[114] I. García-Silva, M. Olvera-Sosa, B. Ortega-Berlanga, V. Ruíz-Rodríguez,
G. Palestino, S. Rosales-Mendoza, Synthesis and characterization of innova-
tive microgels based on polyacrylic acid and microalgae cell wall and their
potential as antigen delivery vehicles, Pharmaceutics 15 (2023) 133.
[115] E.M. Camacho, B. Mesa-Pereira, C. Medina, A. Flores, E. Santero, Engineering
Salmonella as intracellular factory for effective killing of tumour cells, Sci. Rep.
6 (2016) 30591.
[116] V. Balasubramaniam, R.D.N. Gunasegavan, S. Mustar, J.C. Lee, M.F.M. Noh, Isola-
tion of industrial important bioactive compounds from microalgae, Molecules 26
(2021) 943.