Advances and Challenges in Retinoid Delivery Systems in Regenerative and Therapeutic Medicine

REVIEW ARTICLE
https://doi.org/10.1038/s41467-020-18042-2 OPEN
Advances and challenges in retinoid delivery

systems in regenerative and therapeutic medicine
Raquel Ferreira1, Joseph Napoli2, Tariq Enver3, Liliana Bernardino1 ✉ &
Lino Ferreira 4,5 ✉
1234567890():,;
Retinoids regulate a wide spectrum of cellular functions from the embryo throughout
adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits
make retinoids very attractive molecules for medical purposes. In light of some of the phy-
sicochemical limitations of retinoids, the development of drug delivery systems offers several
advantages for clinical translation of retinoid-based therapies, including improved solubili-
zation, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In
this Review, we discuss advances in preclinical and clinical tests regarding retinoid for-
mulations, specifically the ones based in natural retinoids, evaluated in the context of
regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations
of retinoid formulations, as well as prospects to push the field forward, will be presented.
R
etinoic acid (RA) signaling is one of the most important biological pathways in nature,
triggered by RA interaction with nuclear receptors that control gene expression. The
chemical structure of retinol (vitamin A, a RA precursor) was first described by Paul
Karrer in 19311, who was awarded a Nobel Prize in 1937 for the discovery. The use of RA for
skin disorders2 and cancer treatment (acute myeloid leukemia (AML)3 and cervical neoplasia4,5)
started in the 1960s and 1980s (Fig. 1). By the 2000s, RA had been incorporated in many tissue
engineering scaffolds as a stem cell differentiation agent6,7. Over the last 10 years, many dis-
coveries related to the biological role of RA in controlling the biology of hematopoietic stem
cells8,9, tumor-initiating cells10–12, immune cells13, intestinal mucosa wound repair14, cancer
resistance15, and cell reprogramming and differentiation16,17, have further stimulated the interest
in this drug for many other biomedical applications. This interest is confirmed by more than 50
active clinical trials (according to ClinicalTrials.gov) evaluating the effect of RA in cancer (28
trials), mostly in hematological (16 trials) and brain tumors (8 trials), skin pathologies (e.g., acne,
photoaging, eczema) (5 trials), and in other conditions such as inflammation, olfactory loss, and
neuropsychiatric diseases (Table 1).
Clinical applications of RA have highlighted three main limitations of its pharmacological
use. First, RA is poorly soluble in aqueous solutions18 and photosensitive19, which makes
its administration challenging. Secondly, RA induces irritation when applied onto the skin
and increases its catabolism, when it is administered intravenously, reducing its therapeutic
efficacy20. Lastly, RA is involved in many biological processes and thus the systemic delivery
of RA causes side effects. All these limitations motivated researchers to synthesize novel and
1 Health Sciences Research Centre (CICS-UBI), Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal. 2 Nutritional Sciences and
Toxicology, University of California, 231 Morgan Hall, MC#3104, Berkeley, CA 94720, USA. 3 UCL Cancer Institute, University College London, London, UK.
4 Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal. 5 Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
✉email: libernardino@fcsaude.ubi.pt; lino@uc-biotech.pt
NATURE COMMUNICATIONS | (2020)11:4265 | https://doi.org/10.1038/s41467-020-18042-2 | www.nature.com/naturecommunications 1

REVIEW ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18042-2
1980 1983 2013/ 2017

1962 Study describing the First report of the clinical Demonstration of the RA
RA formulation for skin effects of RA in use of ATRA in cancer role in hematopoietic stem
disorders (Ref. 2) leukemic cells (Ref. 3) patients (Ref. 4) cell biology (Ref. 8 and 9)
1971 1982 2010

FDA approved RA First RA controlled delivery RA formulation to control
formulation for acne system (Ref. 134) adult neural stem cells
treament (Ref. 2) activity (Ref. 44)
Fig. 1 Milestones in RA formulations research. ATRA all-trans retinoic acid, FDA Food and Drug Administration, RA retinoic acid.
Table 1 RA formulations tested in past and ongoing clinical trials.
Drug Indication Clinical trial

ATRA-containing liposomes Acne Phase I/II (NA)71
ATRA-containing collagen sponge Mild/moderate intraepithelial cervical neoplasia Phase II (NA)4,5
ATRA + pembrolizumab Advanced melanoma Phase I/Ib (NCT03200847)
ATRA + ipilimumab Advanced melanoma Phase II (NCT02403778)
13-cis RA + cabozantinib Solid tumors Phase I (NCT03611595)
13-cis RA + temozolomide + thiotepa + carboplatin Brain tumor Phase II (NCT00528437)
13-cis RA + 3F8/GM-CSF Neuroblastoma Phase II (NCT01183429)
13-cis RA + 3F8/GM-CSF Neuroblastoma Phase II (NCT01183897)
13-cis RA Neuroblatoma Phase I/II (NCT03291080)
13-cis RA + dinutuximab + lenalidomide Neuroblastoma Phase I (NCT01711554)
13-cis RA + several drugs Neuroblastoma NA (NCT01526603)
ATRA Cholangitis, sclerosing Phase II (NCT03359174)
ATRA + arsenic trioxide APL Phase II (NCT01404949)
ATRA + arsenic trioxide APL Phase III (NCT02339740)
ATRA + arsenic trioxide + gemtuzumab ozogamicin APL Phase II (NCT01409161)
ATRA + idarubicin APL NA (NCT01064557)
ATRA + arsenic trioxide + realgar-Indigo naturalis formula APL Phase III (NCT02899169)
ATRA + several drugs APL Phase IV (NCT02200978)
ATRA + several drugs APL Phase III (NCT02688140)
ATRA + several drugs APL Phase III (NCT00482833)
ATRA Acne vulgaris Phase IV (NCT02620813)
ATRA Multiple myeloma Phase I/II (NCT02751255)
ATRA + rituximab Immune thrombocytopenia Phase II (NCT03304288)
ATRA + 5-azacitidine + lupron Prostate cancer Phase II (NCT03572387)
ATRA Olfactory loss NA (NCT03574701)
ATRA + tranylcypromine + cytarabine AML Phase I/II (NCT02717884)
ATRA + tranylcypromine AML Phase I (NCT02273102)
ATRA + gemtuzumab ozogamicin AML Phase III (NCT00893399)
ATRA + decitabine + cytarabine + G-CSF AML Phase II (NCT03356080)
ATRA + arsenic trioxide + cytarabine AML Phase I/II (NCT03031249)
ATRA + pioglitozone + azacitidine AML Phase II (NCT02942758)
ATRA + gemcitabine + Nab-paclitaxel Pancreatic cancer Phase I (NCT03307148)
ATRA + INCB059872 + azacitidine + nivolumab Advanced malignancies Phase I/II (NCT02712905)
13-cis RA + vorinostat + temozolamide Glioblastoma Phase I/II (NCT00555399)
Retinol + bakuchiol Photoaging; wrinkles Phase I/II (NCT03112863)
9-cis RA + cyclosporine A Hand eczema Phase III (NCT03026946)
AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; GM-CSF, granulocyte-macrophage colony-stimulating factor; NA, not available.
better tolerated synthetic retinoid compounds and to develop applications (cancer and metabolic disease)29, no study has fully
retinoid delivery formulations based on gels, liposomes, micro- covered the application of retinoid formulations for diverse
particles, nanoparticles, and micro-/nanofibers, which in some therapeutic and regenerative medicine applications. In this
cases have been modified to target-specific tissues and cells of review, the role of retinoid formulations in the context of the
interest21. brain, skin, immune system, and cancer applications will be
Recent developments in the use of retinoid for cancer discussed. For each application, the pathological context will be
treatment22,23 and differentiation studies using stem/progenitor briefly presented as well as the effect of retinoid that was admi-
cells8,24, as well as the development of more advanced formula- nistered without any controlled release system. The reasons
tions to control retinoid bioactivity25,26, make this review timely. behind the development of retinoid formulations in each appli-
In addition, except for a limited number of reviews, with a cation will be presented, along with their benefits and limitations,
restricted scientific scope in retinoid formulations (treatment of considering retinoid solubility, photostability, biocompatibility,
skin disorders)27 or highlighting the importance of retinoids in release profile, tissue/cell availability and targeting, and ther-
development (early organogenesis)28 or for specific therapeutic apeutic efficacy.
2 NATURE COMMUNICATIONS | (2020)11:4265 | https://doi.org/10.1038/s41467-020-18042-2 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18042-2 REVIEW ARTICLE
Table 2 Synthetic retinoids in terminated or active (in bold) clinical trials and approved for commercialization.
Drug Receptor activity Indication Clinical trial

Tamibarotene (or Am80) RARα agonist Crohn’s disease Phase II (NCT00417391)
APL Phase II (NCT00520208)
Advanced nonsmall cell lung cancer Phase I (NCT01337154)
AML or myelodysplastic syndrome Phase II (NCT02807558)
Palovarotene RARγ agonist Eye dry disease Phase I (CTP300302)
Fibrodysplasia ossificans progressive Phase III (NCT03312634)
Multiple osteochondromas Phase II (NCT03442985)
Trifarotene (cream) RARγ agonist Moderate facial and truncal acne vulgaris Phase III (NCT03915860)
Autosomal recessive ichthyosis with Phase II (NCT03738800)
lamellar scale
Early cutaneous T-cell lymphoma Phase I (NCT01804335)
Bexarotene (capsules) Pan agonist Refractory cutaneous T-cell lymphoma Approved by FDA since 1999
Tazarotene (gel or cream) Pan agonist Hand–foot skin reactions Phase II (NCT04071756)
Facial acne vulgaris Approved by FDA since 1997
Plaque psoriasis Approved by FDA since 1997
Adapalene (solution, cream, and lotion) Pan agonist Acne Approved by FDA since 1996
AGN194204 RXR agonist Prostate cancer Phase II (NCT01540071)
UAB30/9-cis-UAB30 RXR agonist Nonmelanoma skin cancer Phase I/II (NCT03327064)
Fenretinide (oral powder and intravenous liquid Atypical retinoid Peripheral T-cell lymphoma Phase II (NCT02495415)
emulsion)
Solid tumor (relapsed malignancies) Phase I (NCT01553071)
High risk cancer Phase III (NCT01479192)
Prevention of bladder cancer Phase III (NCT00004154)
Cervical neoplasia Phase III (NCT00003075)
Schizophrenia Phase III (NCT00534898)
Breast cancer Phase III (NCT01357772)
AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; FDA, Food Drug Administration; RAR, retinoic acid receptor; RXR, retinoid X receptors.
Retinoid chemistry and general overview of RA signaling biology and the several formulations developed over the last years
Retinoid chemistry. Retinoids are a class of compounds com- for the delivery of RA.
posed of three regions: a hydrophobic, a central polyene, and a
polar (usually a carboxyl group)29. There are three natural reti-
noids: all-trans RA (ATRA), 9-cis RA (alitretinoin), and 13-cis RA RA signaling. RA is the main biologically active metabolite of
(isotretinoin). To decrease the toxicity of the natural retinoids as vitamin A28. In humans, the only source of vitamin A is obtained
well as to increase their stability and selectivity against a specific through diet, as lipophilic retinol (or its more stable form, retinyl
RAR subtype, several semi-synthetic and synthetic retinoids ester) or as carotenoids. The transport of these retinoids to cells
(including “atypical retinoids”) have been developed29,30. Some of occurs when blood-circulating retinol is bound to retinol-binding
them have been tested in clinical trials and approved for therapy protein (RBP) 4 (Fig. 2). This complex interacts with membrane
(Table 2). At least four molecules have reached phase 4 clinical transporter and receptor stimulated by retinoic acid 6 facilitating
trials: bexarotene, tazarotene, adapalene, and trifarotene29–31. entry into the cytoplasm, where retinol binds to Crbp1 (encoded
Bexarotene has optimal RXR binding, which effectively causes by RBP1). A two-step process converts retinol into ATRA. RA
cancer cell death, particularly in cutaneous T-cell lymphoma. binds to cellular retinoic acid-binding proteins (CRABP), assist-
However, high levels of serum aminotransferase and liver injury ing autocrine and paracrine signaling34. The mechanisms
have been reported32. Tazarotene and adapalene formulations underlying paracrine signaling remain unclear while autocrine
have high affinity and selectivity for RARβ and RARγ, although signaling requires CRABP2 for nuclear entry28,34. In the nucleus,
the first one showed more toxicity than the second in dermato- RA triggers gene transcription by binding to heterodimers formed
logical applications33. Trifarotene is a selective RAR-γ agonist, by RA receptors (RARα, RARβ, and RARγ) and retinoid X
approved in late 2019 in the USA, for the topical treatment of receptors (RXRα, RXRβ, and RXRγ). RAR–RXR heterodimers
acne in patients as young as 9 years old31. Despite the progress interact with a deoxyribonucleic acid sequence known as the
made in the last years in the synthesis of novel retinoids to retinoic acid-response element35, which facilitates the binding of
increase natural retinoid efficacy while reducing their toxicity, it is co-activators to histone acetylase, ultimately leading to the tran-
evident that natural retinoids are still under intense scrutiny as scription of target genes (Fig. 2). Recent data showed that RXRα
demonstrated by a considerable number of clinical trials homodimers can also regulate gene transcription36. Another
(Table 1). The reasons are likely combinatorial: RA is a natural isomer of RA, isomer 9-cis RA (alitretinoin), binds to retinoid X
drug, blocks multiple disease signaling pathways12, in opposition receptors28,35, while 13-cis RA (isotretinoin), has negligible affi-
to some synthetic retinoids that are very selective to a single nity for retinoic acid receptors (RAR or RXR) or cellular RBP37.
receptor-mediated signaling pathway, and it has been used for However, 13-cis RA may be converted into molecules that act as
many years in combinatorial therapy and thus well known by agonists for nuclear RAR and RXR. Importantly, RAR also reg-
clinicians. Because it would be difficult to cover all advances made ulates nonnuclear and nontranscriptional effects, namely the
in retinoid delivery systems in a single review, the authors have activation of kinase signaling pathways38 (Fig. 2). Finally, RA is
chosen to cover only natural retinoids in the present paper. This catabolized by monooxygenases of the cytochrome
focused approach is also supported by recent progresses in RA P450 superfamily34.

a
O R OH O
O
Retinyl esters All-trans retinol All-trans retinaldehyde
(Vitamin A) (Retinal)
O OH
O OH
OH
O
All-trans retinoic acid 9-cis retinoic acid 13-cis retinoic acid
(Tretinoin) (Alitretinoin) (Isotretinoin)
Vas Retinol
cula RBP4
ture
STRA6
STRA6
Retinol Retinyl
Retinol
RAR
RXR
CR Ester
BP
RA RARE
Repression of transcription
CYP26 ADH/RDH
RA
RAR
RA
RXR
Retinaldehyde
P P
RALDH RARE
Degradation Activation of transcription
RA
RAR
Gαq
MAPK
N u c le u s
m
s
Cyt o p la
Non-target cell RA producing cell RA target cell
Fig. 2 Retinoid chemical structures (a) and RA signaling pathway (b). Blood-circulating retinol is internalized through membrane transporter and receptor
stimulated by retinoic acid 6 (STRA6) and converted into all-trans retinoic acid (RA), which binds to cellular retinoic acid-binding protein type 2 for signaling in the
nucleus. RA triggers gene transcription by binding to RA receptors (RAR) and to the retinoid X receptor (RXR). In the presence of the ligand, RAR and RXR
heterodimerize on retinoic acid-response element (RARE) sequences located in promoter regions inducing the transcription of target genes. Of note, RXR may
also homodimerize and trigger gene transcription (not depicted in the illustration). RA signaling may also occur via activation of receptors associated with lipid
rafts located on the cell surface, which trigger transcriptional activation of target genes by histone and receptor phosphorylation in the cell nucleus. ADH alcohol
dehydrogenase, CRBP cellular retinol-binding protein 1, CYP26 cytochrome P450 family 26, Gαq Gq protein alpha subunit, MAPK mitogen activated protein
kinases, P phosphorylation, RALDH retinaldehyde dehydrogenase, RBP4 retinol-binding protein 4, RDH retinol dehydrogenase.
Formulations for the delivery of RA cationic polymers (e.g., poly(ethyleneimine) (PEI))43,44, by phy-
RA has very low solubility in aqueous solution (0.21 μM at pH sical encapsulation in polymeric25,44 or inorganic45 nanoparticles,
7.3)18 and thus requires specific binding proteins (e.g., CRABPs) microparticles46, micelles47,48, liposomes49, or films43, by cova-
to be transported within cells to act at nuclear receptors. In lent attachment of RA to a carrier50, or by immobilization of RA
addition, it has a short (few hours) lifetime, due to its degradation on surfaces of nanoparticles45, among others. Despite several
by a cytochrome P450-dependent monooxygenase system39. reports on RA delivery systems based on polymeric scaffolds or
Moreover, it induces undesirable side effects (congenital mal- electrospun fibrous meshes, the most common strategy has been
formations40 as well as mucocutaneous dryness, headache, and based on liposomal or polymeric nanoparticles formed by
hypertriglyceridemia41) when administered at high concentra- polyesters, polyimines, polysaccharides, and proteins (Table 3). A
tions. Therefore, for more than 35 years, several groups have significant number of formulations have allowed the RA encap-
developed different RA delivery systems to overcome these lim- sulation in the core of the nanoparticles. This RA encapsulation
itations. Delivery systems based in polymeric scaffolds (e.g., was either obtained by (1) physical or (2) chemical interaction
hydrogels, nanofibers), nanoparticles (liposomes, micelles, poly- with the components of the nanoparticle or (3) by physical
meric, dendrimers), microparticles, among others, are described entrapment. Regarding physical interaction, RA is complexed
and summarized in Table 3. with positively charged polymers such as chitosan, or PEI43,51.
The carboxylic acid of RA interacts electrostatically with the
Type and strategies for the delivery of RA. Several strategies amine group located in the polymer, forming a complex that can
have been used to prepare RA delivery systems: namely, by be stabilized by addition of a polyanion and divalent ions43,44.
complexation of RA with proteins (e.g., transthyretin)42 or Concerning chemical interaction, RA is typically conjugated

Table 3 Examples of formulations for the controlled release of RA in the context of cancer, brain diseases, skin diseases, immune
diseases, stem cell differentiation, among other applications.
Type Diameter Loading (μg/mg of carrier) Delivery Applications Ref.

NPs ≃500 nm ≃76 Up to 3 h in the presence of trypsin Pharmaceutical 94
NPs <200 nm 154 Release up to 10 days Cancer 51
NPs 170–230 nm 20 80% cumulative drug release in 4 days Immune diseases 58
NPs 170–185 nm 275 Sustained drug release observed over 72 h Cancer 56
NPs ≃200 nm 86 17% cumulative RA release for 21 days Stem cells and brain diseases 24,44,66
NPs 160 nm 150 50% release after 10 min irradiation Cancer and brain diseases 25,60
NPs ≃100 nm 68–87 5–100% release of RA in 5 days Stem cells and brain diseases 45
NPs ≃214 nm 30 38% cumulative release of RA in 48 h Cancer 120
MPs ≃5.6 μm 80 Pseudo-zero order release for 5 weeks Pharmaceutical 61
MPs ≃8 μm 3 Release up to 10 days Stem cells 7
MPs 190 μm 57 Less than 3 h Cancer 134
Mic 100–500 nm 40–130 NA Pharmaceutical 48
Mic 100–400 nm 2.6 Parenteral administration; <5% in 3 days Cancer 63
Mic 50–200 nm 80% (w/w) 1-month delivery Cancer 47
Lip NA NA 4.4 μg/mL blood per day (in humans) Cancer 49,70,95
EFM 0.95–1.89 μm 5 80% cumulative RA release in 3.5 months Stem cells 135
EFM <5 μm ≃10 ≃9.0 μM for 1 h Stem cells 73
Scaffold NA ≃20 Release over 8–28 h Regenerative medicine 136
Scaffold 0.1–0.85 μm fibers ≃0.3–7 Sustained release of RA for up to 1 week Stem cells 137
EFM, electrospun fibrous mesh; h, hour; Lip, liposome; Mic, micelles; MPs, microparticles; NA, not available; NPs, nanoparticles; min, minute.
chemically to one of the components of the nanoparticle by and macropinocytosis25. Formulations that escaped the endoly-
biodegradable ester50,52, amide26,53–55, or disulfide56 linkages. sosomal compartment accumulated in the cytoplasm in less than
These bonds are susceptible to degradation during specific pH 24 h25,45. RA released in the cell cytoplasm may bind to transport
conditions, in the presence of proteases, or reducing agents that proteins such as cellular retinoid-binding protein II (CRABP-II)
lead to RA release. Concerning physical entrapment, RA is cap- and/or fatty acid-binding protein 5, followed by its transportation
tured during nanoparticle formation57,58 or in the pores of the to the cell nucleus65. The intracellular concentration of for-
nanoparticles59. In a relatively low number of formulations, RA mulations containing RA was dependent on the initial formula-
was immobilized not in the core but on the surface of the tion loading, type of formulation, and type of cell25,45. Uptake
nanoparticle22; however, the concentration of RA immobilized on between 25 and 80 pg of nanoparticles containing RA per cell has
the surface is lower than in the core. Formulations with high been described25.
(above 100 μg/mg of formulation)51,56,60, medium (between 100 The capacity of RA-containing formulations to cross biological
and 25 μg/mg of formulation)44,45, and low (below 25 μg/mg of barriers such as the blood-brain barrier (BBB) (relevance for the
formulation)7,58 loading have been described. treatment of brain cancer and neurodegenerative disorders) is a
The release profile of RA depends on several parameters of the topic largely unexplored. In most cases, the formulations have
formulation, including the size, composition, initial concentration been administered by stereotaxic and not intravenous injection66.
of loaded RA, and its degradation profile. The sustained release of Experimental data in humans indicate that ATRA administered
RA for more than 1 month can be accomplished by encapsulating orally is not able to accumulate in the cerebrospinal fluid67.
it into biodegradable microspheres and tuning the release rate by Clinical trials such as NCT00528437 (Table 1) and others are now
adjusting polymer composition in the formulation61, by the investigating the pharmacokinetics of 13-cis RA and its
encapsulation in polyion complex micelles62 and adjusting accumulation in the cerebrospinal fluid.
polymer composition or drug content, or by the encapsulation
in liposomes49. In general, formulations with high RA loading Preclinical and clinical uses of RA formulations. Formulations
show a slower release profile of the drug51. Because of the containing RA have been used both in preclinical and clinical
hydrophobicity of RA, the occurrence of a burst release in most trials (Fig. 3). Most preclinical tests were performed in mice in the
formulations is negligible44,47,51,63. context of regenerative medicine45,60,66,68 and cancer22,25,56. In
Recent developments to improve the delivery of RA have led to vitro tests showed that ATRA-containing liposomes69 or ATRA-
the design of formulations that can be controlled remotely containing polymeric nanoparticles25,44 were 100–1000 times
(temporally and spatially) by an external stimulus such as light, more active than soluble ATRA in cultured tumor cells25,69 or
ultrasound, or magnetic forces64. These stimuli-responsive neural stem cells (NSC)44. In vivo tests showed that rats49 or
biomaterials are suitable for controlling the kinetic of RA human patients70 treated with ATRA-containing liposomes by
delivery. In that sense, light-activatable nanoparticles containing intravenous administration had no decrease in plasma ATRA
RA that disassemble in minutes after activation by a blue laser at levels while animals/humans treated with oral formulation of
405 nm have been prepared25,26,60. Importantly, these formula- ATRA (nonliposomal) had a significant decrease in plasma
tions presented higher activity than formulations in which RA ATRA levels41,49. The results indicate that the hepatic metabo-
was released by passive diffusion (not light triggered) because lism of ATRA encapsulated in liposomes was inferior to the one
they rapidly saturated nuclear receptors. observed in ATRA administered orally. Both formulations were
safe in human trials. The clinical application of RA-containing
Ability of RA formulations to cross biological barriers. The formulations can be divided into two groups: (1) topical and (2)
cellular uptake of some RA-containing nanoparticles has been oral administration. For topical administration, seven RA-
demonstrated to take <12 h44 by clathrin-mediated endocytosis containing formulations have reached the market for the

Stem
cells
↑ Cell differentiation
↓ Cell proliferation
Skin
↓ Desquamation and erithema

↓ Skin irritation
↓ Inflammation
Nanoparticles
• Anti-microbial activity
Liposomes
Microspheres
Polymeric mycelles
Immune
system
Cancer cells
↓ Inflammation
↓ Migration and invasiveness
↑ Cell death
↑ Cell differentiation
↓ Cell proliferation
Brain
↑ Cell survival (PD, stroke)
↑ Cell differentiation (AD, PD)
↓ Inflammation (stroke)
↓ Memory deficits (AD)
Fig. 3 Main outcomes of RA-based therapies in pathological contexts. AD Alzheimer’s disease, PD Parkinson’s disease, RA retinoic acid, ↑ = increased
effect, ↓ = decreased effect, ● = regulatory effect.
treatment of skin-related diseases (Table 4). Here, current pro- differentiation and tissue formation resembling the phenotype
gresses are concentrated in reducing the toxicity of RA (e.g., by and structure of early human embryos7. Initial studies have used
the use of synthetic retinoids31), and exploring the combination RA as a potent regulator of neural differentiation72. RA down-
of RA with other drugs (Table 4). For oral administration, four regulates expressions of geminin and zinc finger protein Zic2,
formulations containing RA have reached the market, particularly SoxB1 (Sox-1, Sox-2, Sox-3), and Notch-1, which maintain neural
for the treatment of skin diseases (three of them) and cancer (only progenitor cell proliferation. By halting proliferation, RA shifts
one) (Table 4). Others were tested in clinical trials but did not signaling toward differentiation. Several platforms have been used
reach the market. For example, a liposomal-based formulation of for RA delivery alone or in combination with other agents. For
RA (monotherapy) was successfully tested in patients with facial example, RA-containing electrospun fibrous meshes and scaffolds
acne71 and refractory hematological malignancies in a phase II reportedly have an improved effect on stem cell dynamics.
clinical trial70. In the last application, the remission rate (67% Electrospun poly(ε-caprolactone) fibers were loaded with ATRA,
clinical remission) was lower than that obtained using a combi- which led to extremely high local concentrations of this agent and
natorial therapy (77% clinical remission), which included oral the differentiation of murine embryonic cells, within the multi-
administration of ATRA plus idarubicin and thus the liposomal layered scaffolds73. Besides the neurogenic potential of RA, recent
formulation was not further evaluated. Unfortunately, the lipo- studies have highlighted the critical role of RA in the derivation of
somal formulation of RA was not tested in combination with embryonic hematopoietic cells9, lymphoid organs13, and Lan-
other drugs, as it is now being investigated with nonliposomal RA gerhans cells that reside specifically in the epidermis16. Particu-
formulations (Table 1), and this deserves further investigation in larly, ex vivo activation of RA signaling in hemogenic
the near future. endothelium, a small subpopulation of endothelial cells that can
differentiate into hematopoietic cells, increased its transition into
RA delivery systems for regenerative medicine a pool of hematopoietic stem cells. Conversely, RA pathway
Embryonic stem cells. One of the initial applications of RA for shutdown terminated this process9. In addition, fetal type 3
regenerative medicine was as a differentiation agent during innate lymphoid cells (ILC) are modulated by RA signaling in
embryogenesis. The spatiotemporal release of RA by polymeric utero and therefore depend on appropriate maternal dietary
microparticles incorporated within embryonic bodies, derived intake of retinoids throughout pregnancy. This period also
from human embryonic stem cells, was reported to induce cell determines the ability of ILC progenitors to differentiate into

Table 4 Marketed RA formulations.
Name/company Composition Indication Year Ref.

approved
9-cis RA (brand name: Panretin)/ Topical formulation; 0.05% or 0.1% Cutaneous Kaposi’s sarcoma; treatment of 2000 138,139
Eisai Inc. gel containing alitretinoin recalcitrant chronic hand dermatitis
13-cis RA (brand name: Accutane)/ Topical formulation; 0.05% and Photoaging and acne 1982 139
Roche 0.1% cream
ATRA Topical formulation; 0.05% cream Photoaging and acne 1971 2
Gel microsphere formulation containing Topical formulation; macroporous Acne 1997 75,140
ATRA/Advanced Polymer Systems beads, 10–25 μm in diameter
9-cis RA Topical formulation; 0.1% gel AIDS-related Kaposi’s sarcoma 1999 75
Retinol Topical formulation; 0.5–5% Cosmetic NA 75
lotion, cream
Retinaldehyde Topical formulation: 0.01, 0.015, Cosmetic NA 75
0.1% cream
ATRA (brand name: Vesanoid)/Roche Oral formulation of tretinoin Acute myeloid leukemia, in particular, acute 2000 132
promyelocytic leukemia
Acitretin Oral formulation Psoriasis, disorders of keratinization 1997 141
13-cis RA Oral formulation Severe acne/related disorder 1982 75
Retinol Oral formulation Prevent/treat hypovitaminosis A NA 75
AIDS, acquired immunodeficiency syndrome; ATRA, all-trans retinoic acid; NA, not available.
mature lymphoid tissue-inducing cells13. It is possible that some applications, the biological effect of ATRA includes: (1) mod-
of the RA delivery systems developed so far may be used for ulation of proliferation and differentiation of skin cells; (2) anti-
embryonic immune and hematopoietic stem cell development inflammatory activity75. The existence of several types of recep-
studies. tors and their combinations as heterodimers, as well as the ability
of ATRA to modulate the activity of multiple kinase signaling
Adult stem cells. RA is also an important regulator of adult stem pathways independently of the nuclear activation of RAR and
cells. For example, ATRA antagonizes stress-induced activation of RXR receptors, may explain the diversity of ATRA biological
dormant hematopoietic stem cells by restricting protein transla- actions35. ATRA also induces skin angiogenesis and collagen
tion and oxidative stress8. When mice were fed a vitamin A-free deposition, increases the mitotic activity of inter- and follicular
diet to deplete the RA reservoir, animals suffered, among other epithelium, and reduces melanin production75.
effects, functional impairment of hematopoietic stem cells and
their numbers were unable to recover even after injection with an Type of RA-containing formulations. Conventional formula-
immunostimulant8. In addition, RA-based formulations have tions containing ATRA require multiple applications to maintain
been used as inflammatory modulators of stem cells. For example, the therapeutic effect. Therefore, several formulations have been
human mesenchymal stem cells exposed to ATRA-loaded solid developed to improve the long-term effect of ATRA, to reduce
lipid nanoparticles significantly reduced IL-6 and IL-8 expres- side effects like desquamation and erythema, skin irritation, and
sion74. ATRA-containing nanoparticles have been also developed to increase the stability of RA to light19 (Fig. 4). In this sense,
to deliver RA into NSC niches44. The nanoparticles had a higher formulations including phospholipid-based particles (e.g., solid
effect on neuronal differentiation than solubilized RA both lipid nanoparticles and nanostructured lipidic carriers)2,77,
in vitro and in vivo44,66. The effect was mediated by an increase in polymeric nanoparticles2,27,77,78, or polymers conjugated with
transcription of the pro-neurogenic genes Ngn1 and Mash144,66. RA52 have been developed to overcome these issues. The carriers
The formulation was then modified to remotely disassemble and presented a particle size between 100 and 400 nm, a range of zeta
release RA with spatial and temporal resolution, triggered by potential from neutral (liposomes) to negative (polymeric nano-
exposure to blue light60. A single short pulse of light prompted β- particles, ethosomes, solid lipid nanoparticles, and nanos-
catenin-dependent neuronal differentiation and RARα upregula- tructured lipidic carriers), high entrapment efficiency (above
tion. The combined action of blue light and RA enhanced 65%), high photostability (between two- and threefold higher
endogenous neurogenesis. than commercial tretinoin dissolved in ethanol), moderate to high
skin permeation, high skin tolerance, and moderate to high
RA delivery systems for skin diseases antipsoriatic activity. Some of these formulations are easier to
RA mode of action. Several RA drugs are clinically available for scale-up (e.g., solid lipid nanoparticles) than others77,79. In
dermatological treatments, including ATRA and 9-cis-RA, among addition, some formulations (e.g., ATRA-containing liposomes)
others75. For example, 9-cis-RA encapsulated in a gel is an FDA- improve the local effect of RA in the skin and decrease systemic
approved topical agent for cutaneous Kaposi’s sarcoma75. ATRA adsorption78. Moreover, ATRA-containing formulations
has also been tested in clinical trials for the treatment of the same increased significantly the chemical stability of ATRA during
disease76. This sarcoma is associated with human immunodefi- normal storage conditions (e.g., stability for 1 year at 25 °C) and
ciency virus infection and is characterized by a vascular endo- after exposure to UV irradiation (twofold lower photodegrada-
thelioma (i.e., tumor of the endothelial cells). ATRA is used tion)77,80. The protective effect of these formulations was linked
clinically for treating photoaging, acne, and psoriasis. Indeed, to their ability to reflect and scatter UV radiation80. Interaction of
ATRA was approved for acne vulgaris treatment in 19712, and ATRA with a lipophilic amine (stearylamine) decreases ATRA
since then, other drugs (called retinoids because they bind to RAR crystallinity, leading to a formulation with less skin irritating
and/or RXR receptors) have been developed. In these clinical properties77.

Challenges Advances
a Advanced topic formulations b Controlled

improved RA skin RA-release kinetics
a Overcome Retention Skin non-irritative
Agent release
endothelial formulations
barriers
Time
RA
RA RA
Topic formulations do not
reach the circulation,
c Mechanism
avoiding systemic effects of action
b) Limitations in
cell targeting
Intrinsic factors:
ii)
pH
c Delivery of
d Targeting: i)
Temperature
Cell-based delivery &
RA multiple agents
controlled activation Extrinsic factors:
Light
Ultrasound
Fig. 4 Challenges and advances in topical and systemic administration of RA-containing formulations. Challenges include: a crossing endothelial
barriers for extravasation of RA-containing formulations into a specific body region; b targeting of RA-containing formulations to specific cells;
c development of formulations that combine RA with other pharmacological agents, able to release each agent with a specific release kinetics. Advances
include: a development of formulations with less toxicity; b release of RA with variable release kinetics to achieve variable biological actions; c action
mechanism of RA during development and disease; d use of cells to transport RA-containing formulations to specific regions in the body followed by the
triggering of the formulations by intrinsic (e.g., temperature, pH) or extrinsic (e.g., ultrasound, light) stimuli. RA retinoic acid.
Preclinical and clinical applications. RA-containing formula- can act as transporters of formulations that will be activated by
tions are under clinical evaluation for the treatment of acnes local (e.g., pH) or remote triggers (e.g., light, ultrasound, mag-
vulgaris, hand eczema and photoaging (Tables 1 and 3). An netism)25,82 once they reach the target site.
ATRA-loaded liposomal formulation has been tested in a pilot
trial71. Patients with facial acne treated with the liposomal for- Alzheimer’s disease. AD is a neurodegenerative disease caused by
mulation showed higher lesion improvements than with con- the accumulation of amyloid-beta peptides, hyperphosphorylated
ventional formulations. The higher efficacy of the ATRA-loaded tau filaments, and brain vascular changes leading to cerebral
liposomal formulation was attributed to enhanced penetration of amyloid angiopathy83. In experimental models of AD, RA: (1)
RA across the stratum corneum. A commercial tretinoin gel inhibited oxidative damage and mitochondrial dysfunction; (2)
microsphere formulation consisting of microspheres with 10–20 increased ApoE expression and suppressed inflammation; and (3)
μm in size75 has reached the market. A common issue in improved learning and memory84,85. Nanoparticles have been
designing controlled RA release systems for skin applications is developed to efficiently deliver ATRA and small interfering RNA
the limited efficacy in terms of cell targeting (Fig. 4). Formula- to promote NSC differentiation in AD45. In this study, the
tions such as micro- and nanoparticles might have the capacity to transplantation of nanoparticle-treated NSCs in AD mice
target particular cells by specificities in their physicochemical improved cognition and memory.
properties or by incorporating in their surface peptides, proteins,
or aptamers that recognize specific cell receptors. Due to their
Parkinson’s disease. PD is characterized by selective degenera-
size, these formulations might accumulate preferentially in some
tion of dopaminergic neurons in the substantia nigra and by the
regions of the skin, such as the follicular adduct and thus act in
accumulation of α-synuclein and Lewy bodies (protein inclusions
those biological environments. Indeed, follicular targeting is
in neurons)68. In experimental models of PD, RA protected
important for the treatment of acne because it increases the
against neurodegeneration of midbrain dopaminergic neurons in
therapeutic effect of retinoids, while reducing their potential side
the substantia nigra86,87. Stereotaxic injection of ATRA-
effects. Polymeric micelles of diblock methoxy-poly(ethylene
containing nanoparticles in the striatum protected nigral dopa-
glycol)-poly(hexyl-substituted lactic acid) copolymer favor folli-
minergic neurons in an in vivo PD model68. Accordingly, RA-
cular aduct-targeted delivery of ATRA81.
containing nanoparticles increased expression of Nurr1 and
Pitx3, key regulators of dopaminergic neuronal development and
RA delivery systems for brain diseases maintenance68,88.
Several formulations containing RA have been used to treat
Alzheimer’s disease (AD), Parkinson’s disease (PD), and stroke Stroke. In the case of stroke, changes in the vasculature, or BBB
(Table 1). Current challenges in the use of RA formulations for permeability or function, may cause or enable the progression of
brain diseases are ascribed to low accumulation in the brain after CNS diseases89. RA/RAR signaling is critical for BBB differ-
intravenous administration (Fig. 4). Although the formulations entiation and integrity90. Recent studies have shown protective
having affinity ligands in their surface have increased retention in effects of ATRA-containing nanoparticles in stroke. The for-
the brain, only a minor fraction of the injected formulation mulation enhanced endothelial cell proliferation and tubule net-
reaches the brain and crosses the BBB. Cell-mediated delivery work formation and protected against ischemia-induced death in
(e.g., T cells, monocytes) may be used to overcome this issue: cells endothelial cell lines and endothelial progenitor cells isolated

from ischemic stroke patients24. Moreover, when intravenously nanoparticles with the surface of hematopoietic stem cells102, or
injected, RA formulations restored neuronal and vascular func- by surface modification of nanoparticles with antibodies (e.g.,
tions in a prenatal model of brain ischemia91. CD45.1, CD117)103,104 or aptamers (E-selectin thioaptamer)105.
Recently, we have developed a new platform to target bone
marrow, based on light-activatable nanoparticle formulations
RA delivery systems for the treatment of cancer containing ATRA25,26. The formulation was highly internalized
Blood cancers. RA mode of action: one of the first applications of by leukemia-initiating cells and accumulated in the cell
RA delivery systems was for the treatment of blood cancers. The cytoplasm. Once leukemia-initiating cells transfected with light-
antitumor activity of ATRA was demonstrated in 1980 in acute activatable nanoparticles containing RA were administered
promyelocytic leukemia (a subtype of AML accounting for 5% of intravenously in leukemic mice they tend to home in the bone
AML cases)3. Since then, many clinical trials have evaluated the marrow, in the proximity of other leukemia cells. The irradiation
antitumoral efficacy of the drug alone or in combination with of bone marrow with a blue light induced photo-disassembly of
arsenic trioxide or idarubicin92. RA promoted terminal differ- the nanoparticles within the cells and consequently their
entiation of leukemic cells while reducing their proliferation. The differentiation. These cells then secreted extracellular vesicles
antitumoral activity of RA (enhanced by cooperation with arsenic containing ATRA that interfered with cells supporting the stem
trioxide)11 was due to inhibition and degradation of prolyl iso- cell niche.
merase Pin1, which has a critical role in coordinating multiple Preclinical and clinical applications: most RA-containing
phosphorylation events during oncogenesis12. Therefore, RA has formulations are under clinical evaluation for the treatment of
the unique property of blocking multiple cancer-driving pathways blood tumors (Table 1). Current challenges for translation of
simultaneously. Unfortunately, the therapeutic efficiency of RA formulations to the clinic are related to two factors: (1)
ATRA-based therapies remains to be demonstrated in patients capacity to incorporate multiple drugs besides RA; (2)
with AML without acute promyelocytic leukemia. efficiently target hematopoietic cells (Fig. 4). In most cases,
Type of RA-containing formulations: physicochemical proper- RA-based monotherapies are less efficient in interfering with
ties as well as release properties of RA-containing formulations tumorigenesis and cancer progression than combination
for the treatment of blood cancers are summarized in Table 3. therapies29. The efficiency of combination therapies is likely
With the exception of some formulations for parenteral ascribed to several factors: (1) a combination of drugs may act
administration63 or for intracellular delivery in ex vivo at different sites of the antiproliferative signaling pathway and
conditions25,26, most RA-containing formulations have been thus be more effective in the inhibition process; (2) combina-
developed for intravenous delivery. These formulations were tion therapies may decrease the probability of cancer resistance;
based in liposomes49,70,93, microspheres61, polymeric micelles63, (3) synergies between the drugs reduces the dose necessary for
and nanoparticles50,94. In most cases, ATRA was encapsulated in therapy, as well as their toxicity and treatment time. Currently,
the formulation49,70, or chemically conjugated to the carrier50. several clinical trials are investigating the combination of
This effort was motivated by the fact that some of the patients ATRA with arsenic trioxide, with arsenic trioxide and
treated with ATRA relapsed during the 4–6 weeks treatment of gemtuzumab ozogamicin (a monoclonal antibody against
daily oral administration. Follow-up studies indicated that CD33 conjugated with ozogamicin, a cytotoxic agent), with
resistance was due to the decrease of ATRA concentration in epigenetic regulators such as tranylcypromine, with decitabine,
the plasma, and thus the inability of the drug to differentiate cytarabine and granulocyte-stimulating factor, among others, in
leukemic cells41. The decrease was attributed to an induction of the context of AML (Table 1). Unfortunately, most RA
ATRA catabolism and increased levels of RA-binding protein20. formulations tested so far for blood cancers have not explored
Several ATRA delivery systems showed relative success in the simultaneous controlled release of RA and other agents.
reducing the induction of ATRA catabolism and thus maintain- Thus, further investigation is needed to address this issue. It
ing RA for longer periods in blood plasma. For example, should be noted that a liposomal formulation having two agents
microspheres of poly(L-lactide) showed a nearly constant release (non-RA drugs) has been approved recently for AML106.
rate of ATRA for 5 weeks61. In addition, intravenous adminis- Another important challenge in clinical translation of RA
tration of RA-containing liposomes for 7 weeks in rats did not formulations is targeting. Preclinical tests have addressed
enhance RA catabolism49. Maintenance of RA in the plasma, via different leukemia cell targets that have not yet been explored
liposomal-encapsulated ATRA, reduced the number of relapses95. in clinical trials. The recent re-approval of gemtuzumab
Targeted delivery of RA-containing formulations to the bone ozogamicin106 may inspire new approaches for RA formulation
marrow has recently attracted much attention96. Bone marrow is targeting.
the residence of hematopoietic stem cells that give rise to myeloid
and lymphoid cell lineages. In leukemia patients, hematopoietic
stem cells are the target of genetic mutations and thus aberrant RA delivery systems for the treatment of solid tumors. RA
activity. These altered cells are difficult to eliminate by mode of action: one of the first applications of RA formulations
conventional antitumoral agents because drugs do not reach the for the treatment of solid tumors was in mild/moderate intrae-
bone marrow at the required concentration, and because the stem pithelial cervical neoplasia4,5. ATRA was released by a collagen
cell niche (a physical and functional entity that supports the self- sponge with a cervical cap at the tumor site by insertion in the
renewal and differentiation of stem cells) changes/adapts after cervix. A dose of ∼0.4% of RA was selected for a phase II trial.
chemotherapy, protecting altered cells97. RA is a potential Fifty percent of the patients showed a total regression of the
treatment, combined with other drugs, for AML98 and chronic disease4,5. Systemic and cervical side effects were mild and vaginal
myeloid leukemia99. In the last 10 years, a new therapeutic side effects were moderate and tolerable. Unfortunately, the for-
paradigm has emerged based on the targeted delivery of mulation did not reach the market, likely because it was not
formulations to bone marrow96. Targeted delivery can be sufficient to reverse or suppress more advanced dysplasia with
achieved by surface modification of nanoparticles with bispho- acceptable local side effects in phase III clinical trial107. The
sphonate to promote binding to bone100, by surface modification antitumoral activity of RA is linked to its differentiation and cell
of liposomes with folate to target the folate receptor, which is growth arrest properties108. RA has been tested in several clinical
highly expressed by AML cells21,101, by the conjugation of trials and the results showed that RA alone did not present

significant antitumoral activity against breast cancer109; however, described as potent anti-inflammatory and therefore protective in
when RA was combined with tamoxifen or paclitaxel it showed pathologies such as chronic obstructive pulmonary disease74,
moderate antitumoral activity110,111. rheumatoid arthritis122, psoriasis123, and inflammatory bowel
Type of RA-containing formulations: in the last years, disease124. At a cellular level, RA inhibited IL-6-driven induction
significant effort has been made by the scientific community to of proinflammatory Th17 cells and promoted the differentiation
develop formulations able to release RA and other antineoplastic of Treg cells, which are important to suppress excessive immune
drugs. For example, nanoparticles containing both ATRA and responses125.
paclitaxel (to inhibit cell division because the cell cannot disrupt The role of RA in the gastrointestinal tract is particularly
the polymerized tubulin for cell division) had superior efficacy relevant since it is produced and metabolized in the intestine,
than formulations containing only paclitaxel53. In addition, pH- where it regulates the differentiation and function of diverse
sensitive nanoparticles have been designed to release all-trans immune cells and supports mucosal barrier immunity126. Indeed,
retinal and doxorubicin in weakly acidic tumors (pH 6.5) or ATRA regulates the activity of CD161+-Treg cells that support
acidic intracellular environments such as endosomes/lysosomes wound repair in intestinal mucosa14. These C-type lectin CD161
(pH 4.5–5.5)112. All-trans retinal was chemically conjugated to regulatory T cells were found to induce cytokines that promoted
the nanoparticle polymer by hydrazone bonds that were labile epithelial barrier healing in the gut. Accordingly, several studies
under acidic conditions. Compared to free drugs, the nanoparticle have focused on the impact of vitamin A intake (i.e., RA obtained
formulation increased the accumulation of the all-trans-retinal from the diet), both during development and in the adult.
and doxorubicin at the tumor site and induced higher levels of Accordingly, the levels of retinoids obtained from the maternal
cell senescence and antitumoral activity. Progress has also been diet increase the size of secondary lymphoid organs and the
made in targeting cancer-initiating cells, which are resistant to efficacy of adult immune responses13, while the depletion of
chemotherapy and associated with tumor recurrence. ATRA vitamin A, in the adult, led to a decrease of Th1, Th17, and ILC3
liposomes and nanoparticles encapsulating simultaneously ATRA responses (a synonym of lowered immunity to bacterial
and doxorubicin have been used successfully to arrest the infection), an effect which is counteracted through time127.
proliferation of breast cancer-initiating cells and to differentiate
them113,114. In a combinatorial approach, RA differentiated Type of RA-containing formulations. Several RA formulations
cancer-initiating cells, whereas antineoplastic agents, such as have been developed to target immune cells and induce an
doxorubicin, killed noncancer-initiating cells. immunomodulatory response, such as solid lipid nanoparticles74,
Preclinical and clinical applications: the results of several polymeric nanoparticles58, nanostructured lipid carriers124,
clinical trials indicate that 13-cis RA, alone or in combination, among others. RA formulations with an average diameter of
seems to be effective at different levels against malignant 130–250 nm58,74 and an entrapment efficiency of ATRA between
gliomas115, and cancer occurring in the central and peripheral 2.3 and 310 μg124 per mg of nanoparticle have been developed.
nervous system116. Moreover, ATRA inhibits proliferation of Macrophage phagocytosis was influenced largely by the physi-
glioblastoma cells in vitro and in vivo, while promoting their cochemical properties of nanoparticles. For example, uptake is
differentiation117,118. At high concentrations, ATRA may induce higher in nanoparticles with high negative or positive surface
cell apoptosis119. RA-coated solid lipid nanoparticles showed charges128. In general, RA formulations showed sustained intra-
higher in vitro toxicity relatively to glioblastoma cells than the cellular RA delivery for a few days58,74. RA formulations are
parent drug120. In addition, polymeric micelles composed by taken up by macrophages and induce anti-inflammatory
methoxy-grafted chitosan and encapsulating ATRA were more responses by suppressing NF-kB signaling and increasing bone
effective at inhibiting glioblastoma cell line migration in vitro morphogenetic protein 2 signaling (pivotal for bone and cartilage
than the free agent62. development), as well as by enhancing the production of anti-
The immune system plays an important role in modulating inflammatory cytokines (e.g., IL-10)58,124. In addition, RA for-
tumor progression; however, limited infiltration of immune cells mulations have the capacity to enhance the differentiation of
in the tumor site, as well as the existence of immunosuppressive naïve T cells to regulatory T cells124. The advantages of RA-
agents, hamper their biological role. Therefore, a combination of containing nanoparticles versus free RA were demonstrated by
chemotherapy with immunotherapy might be a better strategy to inhibiting expression of IL-6 and IL-8 in alveolar epithelial cells74,
fight tumor biology. Indeed, immunotherapy may increase the but were not demonstrated in the induction of immune cell
sensitivity of cancer cells to chemotherapy and thus reduce its differentiation.
side effects121. Recently, a chemo-immunotherapy approach for
melanoma was developed based on biodegradable hollow
mesoporous nanoparticles containing three drugs: doxorubicin, Preclinical and clinical applications. Clinical trials using free RA
ATRA, and interleukin (IL)-223. IL-2 is a T-cell growth factor and in the context of immune thrombocytopenia (severe bleeding
can thus facilitate proliferation and activation of T cells. Animals disorder) and sclerosing cholangitis (inflammation and scarring
treated with formulations containing the three agents were the of the bile ducts) are active and will evaluate the role of RA as
ones with the most effective tumor growth inhibition and immunotherapy (Table 1). Current challenges for using RA for-
decreased metastasis. ATRA was effective in differentiating mulations for immune diseases are related to the in vivo
myeloid-derived suppressor cells, and in synergy with doxor- demonstration of RA effects. This requires the use of formula-
ubicin, contributed to increase the number of dendritic cells at the tions able to target-specific cells, particularly immune cells, and
tumor site; IL-2 facilitated the proliferation of CD8+ T cells at the their surface receptors (Fig. 4). There are classes of molecules,
tumor site to promote effective tumor killing. including antibodies, carbohydrates, peptides, aptamers that can
be attached to RA formulations to target more specific immune
cell populations.
RA delivery systems for immune diseases
RA mode of action. Inflammation is essential for the regulation Future outlook
of tissue homeostasis and barrier integrity (e.g., BBB, mucosal The advantages of RA formulations compared to free RA have
barrier). However, when dysregulated, it contributes to the been demonstrated in various biomedical applications. These are
pathophysiology of many diseases. In fact, retinoids have been related to: (1) increased bioavailability; (2) decreased toxicity and

skin irritation; (3) increased in vivo half-life; (4) increased RA formulations requires the use of linkers susceptible to pH or
photostability; (5) increased cell targeting and capacity to cross enzymes present in the targeted cell/tissue. For example, several
biological barriers. The advantages of RA formulations have been RA-polymer conjugates have been developed to release RA by
demonstrated in preclinical (in all applications described in this hydrolytic cleavage of ester bonds50,52, by enzymatic cleavage of
review) and clinical trials (cancer and skin applications)71,129. In amide bonds by proteases present in the targeted tissue28,65–67 or
the case of cancer, recent studies indicate that the antitumoral by reducing environments56 such as the cell cytoplasm. In addi-
activity of RA might be greatly enhanced with formulations that tion, linkers may be cleaved by an external trigger, such as light
extend the in vivo half-life of RA12. with the consequent release of RA25,26.
RA formulations have some limitations that preclude a more RA is an evolutionarily ancient and conserved molecule with
significant translation to the clinic. For example, ATRA, 9-cis-RA, overlapping and pleiotropic effects. Given the diverse pharma-
and 13-cis-RA, which belong to the first generation of retinoids, cological activities of RA, there is ample potential for developing
are being gradually replaced in some applications by third- therapeutic applications. Thus, the fine-tuning of RA-properties
generation retinoids130. Although many of these formulations and targeted formulation-based delivery is very attractive for
have been tested in preclinical models, the clinical translation is customized and more efficient therapies. It is expected that the
still relatively low. For example, although nanoparticles delivered knowledge gathered in the development of retinoid formulations
systemically have been extensively explored in animal tests, inspires others to generate delivery systems based on different
clinical translation remains compromised because of delivery drugs thus expanding the field of regenerative and therapeutic
(e.g., limited efficacy in terms of cell targeting, limited capacity to medicine.
cross biological barriers), technical (e.g., scale-up), and regulatory
aspects (e.g., study design and approval challenges). Use of Received: 20 July 2019; Accepted: 1 August 2020;
antibodies, peptides, aptamers immobilized in the surface of
nanoparticles to target-specific cellular receptors may accelerate
clinical translation of RA formulations. Indeed, several targeted
nanoparticle formulations are now being tested in clinical trials
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Advances and challenges in retinoid delivery

NATURE COMMUNICATIONS | (2020)11:4265 | https://doi.org/10.1038/s41467-020-18042-2 | www.nature.com/naturecommunications 1

1980 1983 2013/ 2017

1971 1982 2010

Table 1 RA formulations tested in past and ongoing clinical trials.

Drug Indication Clinical trial

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Drug Receptor activity Indication Clinical trial

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Type Diameter Loading (μg/mg of carrier) Delivery Applications Ref.

NATURE COMMUNICATIONS | (2020)11:4265 | https://doi.org/10.1038/s41467-020-18042-2 | www.nature.com/naturecommunications 5

↓ Desquamation and erithema

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Table 4 Marketed RA formulations.

Name/company Composition Indication Year Ref.

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a Advanced topic formulations b Controlled

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