Abstract
Hemangioma of infancy is the most common vascular tumor during infancy
and childhood. Despite the proven efficacy of propranolol treatment,
certain patients still encounter resistance or face recurrence. The
need for frequent daily medication also poses challenges to patient
adherence. Bleomycin (BLM) has demonstrated effectiveness against
vascular anomalies, yet its use is limited by dose-related
complications. Addressing this, this study proposes a novel approach
for treating hemangiomas using BLM-loaded hyaluronic acid (HA)-based
microneedle (MN) patches. BLM is encapsulated during the synthesis of
polylactic acid (PLA) microspheres (MPs). The successful preparation of
PLA MPs and MN patches is confirmed through scanning electron
microscopy (SEM) images. The HA microneedles dissolve rapidly upon skin
insertion, releasing BLM@PLA MPs. These MPs gradually degrade within
28 days, providing a sustained release of BLM. Comprehensive safety
assessments, including cell viability, hemolysis ratio, and intradermal
reactions in rabbits, validate the safety of MN patches. The
BLM@PLA-MNs exhibit an effective inhibitory efficiency against
hemangioma formation in a murine hemangioma model. Of significant
importance, RNA-seq analysis reveals that BLM@PLA-MNs exert their
inhibitory effect on hemangiomas by regulating the P53 pathway. In
summary, BLM@PLA-MNs emerge as a promising clinical candidate for the
effective treatment of hemangiomas.
Graphical Abstract
graphic file with name 12951_2024_2557_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s12951-024-02557-7.
Keywords: Hemangioma, Bleomycin, Microneedle, Polylactic acid,
Microsphere
Introduction
Hemangioma of infancy is the most prevalent vascular tumor and the most
common benign neoplasm during infancy and childhood, with historical
incidence rates ranging from 2 to 10% and an overall prevalence of 4.5%
[[45]1]. Hemangiomas are categorized as infantile or congenital.
Infantile hemangiomas (IHs) are characterized by rapid postnatal
proliferation lasting for 3–6 months, followed by slow regression over
years [[46]2]. However, about 10–15% of larger hemangiomas or those in
specific locations, such as the head, neck, perineal, and anal regions,
may lead to complications like ulceration, disfigurement, or
obstruction during the proliferative stage [[47]3]. Unlike IHs, which
typically enlarge after birth and then involute, congenital hemangiomas
(CHs) proliferate in utero and are fully developed at birth. Three
major subtypes of CHs are defined: rapidly involuting congenital
hemangioma (RICH) [[48]4], noninvoluting congenital hemangioma (NICH)
[[49]5] and partially involuting congenital hemangioma (PICH) [[50]6].
RICH undergoes complete involution within the initial 6 to 14 months
whereas NICH expands in proportion to the child's growth without
regression.And PICH regresses incompletely and then stabilizes. CHs may
exhibit persistent ulceration, hemodynamic instability, and
thrombocytopenia [[51]7]. Hence, besides hemangiomas that naturally
regress, active treatments are necessary for hemangiomas that do not
regress and are accompanied by complications.
Currently, three primary treatment methods exist for hemangiomas:
medication, laser therapy, and surgery [[52]8]. Laser therapy and
surgery, although effective, come with a relatively high cost and may
not be suitable for every patient. Consequently, non-invasive,
cost-effective, and convenient oral medications have become the
preferred choice since 2008. Propranolol, a nonselective beta-blocker,
has emerged as the first-line therapy for complicated infantile
hemangiomas [[53]9]. Notably, Hemangeol (propranolol hydrochloride oral
solution) stands out as the only FDA-approved drug with proven safety
and efficacy for treating infantile hemangiomas [[54]10, [55]11].
However, potential adverse effects of Hemangeol include sleep disorders
and aggravated respiratory tract infections [[56]12]. The requirement
for twice-daily oral administration may also reduce patient adherence.
Additionally, 0.9% of reported cases were resistant to propranolol, and
18% experienced recurrence after ceasing oral drugs [[57]13].
Meanwhile, timolol maleate has also been widely studied for topical
application for IH treatment due to the commercial availability of
timolol ophthalmic drops. For both propranolol and timolol, topical
treatment is typically applied multiple times a day, which can reduce
adherence to therapy [[58]14, [59]15].
Bleomycin (BLM), a chemotherapy agent that inhibits DNA synthesis, is
gaining increased prominence in sclerotherapy for patients with
vascular anomalies [[60]16]. Our team has reported the effectiveness of
intralesional bleomycin injection in treating hemangiomas resistant to
propranolol [[61]17]. However, the repeated use of BLM is associated
with potential dose-related complications, such as pneumonitis and
pulmonary fibrosis, significantly limiting its practical application
[[62]18, [63]19]. In contrast, topical dermal application of BLM is
considered a more favorable option for treating superficial
hemangiomas. This method minimizes undesirable effects compared to oral
administration, as it can achieve a high concentration of the drug
locally while reducing systemic exposure. Despite these advantages, the
transdermal transport efficiency of most drugs is hindered by the
stratum corneum, leading to unsatisfactory therapeutic effects in the
majority of cases [[64]20, [65]21]. Consequently, there is an urgent
need to develop effective strategies to improve the efficiency of
topical drug delivery.
Microneedle (MN) patches, which are topical transdermal drug delivery
systems consisting of arrays of micrometer-sized needles and
substrates, have seen significant development in recent years [[66]22,
[67]23]. These microneedles create transport pathways through the
skin's stratum corneum to deliver various therapeutic molecules
[[68]24, [69]25]. Various materials have been designed for MN patches
recently [[70]26]. Non-degradable MN structures generally exhibit
excellent mechanical properties, ensuring the structural integrity of
MNs during use [[71]27]. However, the release of the active
pharmaceutical ingredient (API) from non-degradable MNs can continue
for several days or even weeks, making long-term wear inconvenient and
aesthetically displeasing [[72]24]. Therefore, rapidly biodegradable
(dissolvable) MN patches may be more suitable for treating hemangiomas.
Additionally, the customizability of microneedle size, including the
patch size and the height of the tips, can meet the specific needs of
individual patients in clinical applications.
Hyaluronic acid (HA), a widely used water-soluble biomacromolecule,
exhibits excellent biocompatibility [[73]28]. HA-based MNs achieve
rapid drug delivery by separating the needles from the base [[74]29,
[75]30] and regulating the amount of drug released by controlling the
loading capacity of the drug, leaving no poisonous residue after
insertion [[76]31]. However, simple HA-based MN patches dissolve too
rapidly upon injection into the skin [[77]32], resulting in swift
metabolization and release of the API. This necessitates frequent usage
within a short timeframe, elevating the risk of bacterial infection
[[78]33]. Thus, extending the drug release time with the shortest use
of microneedle patches is a key consideration.
Polylactic acid (PLA), one of the most promising biodegradable
polymers, is derived from renewable natural plants such as corn, wheat,
rice, and sugar cane [[79]34, [80]35]. It can be degraded into water
and carbon dioxide in vitro or low molecular weight PLA and innocuous
lactic acid in vivo. PLA microspheres have been used for the delivery
of different drugs, including small molecule drugs, polymers, and
proteins. With the increasing demand for eco-friendly polymeric
materials in the biomedical field, such as implantable biomaterials and
medicine packages, PLA has become one of the most promising
alternatives [[81]36, [82]37].
To develop a more efficient therapy for treating hemangiomas, we
propose a simple strategy for synthesizing BLM-loaded HA-based MN
patches. BLM was encapsulated during the synthesis of PLA microspheres
(MPs). The successful preparation of PLA MPs and MN patches was
confirmed through scanning electron microscopy (SEM) images.
Furthermore, we investigated the in vitro release profile of BLM from
PLA MPs. The safety of the MN patches was comprehensively validated
through assessments of cell viability, hemolysis ratio, and irritation
and skin sensitization tests on rabbits. Subsequently, we established a
murine hemangioma model and demonstrated the inhibitory effect of
BLM@PLA-loaded MN patches on hemangiomas by modulating the P53 pathway.
Materials and methods
Materials
HA (Mw: 90,000) was purchased from MEILUNE Biomed. PLA, Bleomycin and
polyethylene glycol were obtained from Macklin. CHCl[3] were purchased
from Sigma-Aldrich. Deionized water was used throughout all the
experiments. Polydimethylsiloxane (PDMS) master molds of MNs (320 μm
base diameter, 830 μm height, and 20 × 20 arrays with 650 μm tip-tip
spacing) were provided by Taizhou Microchip Pharmaceutical Technology
Co., Ltd, China.
Characterization
The hemoglobin release was recorded by the OD values at 576 nm tested
by the Eon microplate spectrophotometers (Bio Tek Instruments, Inc.).
Thermogravimetric Analysis (TGA) was performed on TGA 4000 (Perkin
Elmer Co., Ltd) with the temperature ranged from 30 to 800 °C under
N[2]at a heating rate of 10 °C min^−1. The SEM images were examined by
ZEISS Gemini 300 fitted with Oxford Xplore EDS detector. The TEM images
were examined by JEOL2100F fitted with JED2300 EDS detector. Fourier
transform infrared (FT-IR) spectra of the patches were recorded by a
Thermo-Scientific Nicolet 6700 FT-IR spectrometer (4000–400 cm^−1).
X-ray Photoelectron Spectroscopy (XPS) measurements were recorded on
Thermo-Scientific K-Alpha with Al Kα radiation. UV–visible spectroscopy
was tested with SHIMADZU UV-3600i Plus.
Synthesis of PLA and BLM@PLA MPs
The PLA MPs were synthesized as the previous work reported [[83]38].
Blank PLA MPs were prepared through emulsion solvent evaporation method
as shown in Fig. [84]1. PLLA (5 g) particles and Polyvinyl Alcohol
(PVA) were dissolved in CHCl[3] (100 mL) and deionized water (100 mL)
respectively. The PLLA solution was then added to aqueous PVA solution
under magnetic stirring at a rotation speed of 900 rpm. The emulsion
was stirred continuously overnight until the CHCl[3] evaporated
completely. Afterwards, the mixed emulsion was filtrated with a
double-layer sieve mesh (pore diameters were 7.5 μm and 5 μm
respectively) and washed by deionized water to remove the residual PVA.
The PLA MPs which remained between the sieve mesh were freeze-dried and
collected for further use.
Fig. 1.
[85]Fig. 1
[86]Open in a new tab
Schematic of BLM@PLA MPs loaded MN patches controlling drug release and
treating hemangiomas. a The preparation process of MN patches. b The
fabrication process of BLM@PLA MPs. c The illustration of the BLM
release process. The right insert presents the internal environment of
BLM@PLA MPs. d Internal structure diagram of MPs. e The degradation
process of MPs in subcutaneous tissue. f The entire process of applying
BLM@PLA-MNs to treat hemangiomas
The BLM@PLA MPs were prepared via one-step synthesis by mixing BLM
(3 mg) to the PVA solution in the first step of synthesizing PLA MPs
and followed the similar process with PLA MPs.
Preparation of HA-based MN patches
A customized PDMS master mold of MNs (320 μm base diameter, 830 μm
height, and 20 × 20 arrays with 650 μm tip-tip spacing) was used to
fabricate MN patches. The HA aqueous solution (5 wt%, 1 mL) and BLM
(30 mg), PLA MPs (30 mg) or BLM@PLA MPs (30 mg) were ultrasonicated for
30 min and subsequently cast into the PDMS mold until just filling up
the all cavities. Afterwards, the mold filled with solution was
degassed by vacuum oven for 30 min at room temperature, and the mold
was air-dried overnight at room temperature for complete drying and the
processes repeated for 3 times to fully fill all cavities. And then
place the mold in dry environment for further use. Finally, additional
2 mL HA aqueous was cast into the mentioned mold above gradually, and
the mold was placed for further air-drying for 24 h, after which the
patch was carefully peeled from the mold and stored in a desiccator
until use. Specifically, BLM, PLA and BLM@PLA MPs loaded MN patches
were denoted as BLM-MNs, PLA-MNs and BLM@PLA-MNs respectively.
Loading efficiency of BLM in PLA MPs
The loading capacity of BLM could be calculated via thermo-gravimetric
analysis curves of PLA and BLM@PLA MPs. Specifically, after heating
5 mg of BLM, PLA and BLM@PLA MPs from 30 to 800 °C respectively
(heating rate: 10 °C min ^−1), the BLM content in BLM@PLA could be
accurately calculated with the remaining residue. The detailed formula
of drug loading efficiency was presented as follows (A, B and C
represent as PLA, BLM@PLA and BLM respectively):
[MATH: BLM loading rate%=Aresidual
rate-Bresidu
al
rateAres
idual
rate-Cresidu
al
rate×100% :MATH]
In vivo intradermal irritation experiment
All procedures followed were in accordance with the Declaration of
Helsinki. All procedures were reviewed and approved by Shanghai Ninth
People's Hospital Central Lab IACUC (Permit Number: SYXK (Shanghai)
2016-0016), and all experiments conformed to the relevant regulatory
standards. New zealand white rabbit (2.5–3.5 kg) were purchased from
the Shanghai JieSiJie Laboratory Animal Co.Ltd and housed according to
ISO 10993-2 regulations.
In this experiment, samples(HA-MN/PLA-MN/BLM-MN/BLM@PLA-MN) were
extracted using 0.9% saline solution (polar) and sesame oil (Non-polar)
at 37 °C, 60 rpm constant temperature shaking incubator for 72 h.
Negative control solution was prepared under the same conditions.
Eighteen hours before the experiment, thoroughly remove the fur from
both sides of the animal's spine on the back. On the day of the
experiment, 0.2 mL of the polar extracts prepared with 0.9% saline were
injected intradermally at five points on one side of the rabbit's
spine. On the other side of the spine, polar solvent control (0.9%
saline) solution was injected. The non-polar extract and the non-polar
control solution were handled in the same manner. The erythema, edema,
and necrosis of injection sites were observed and recorded immediately,
at (24 ± 2) h, (48 ± 2) h, and (72 ± 2) h after the injections. The
dorsal skin of rabbit was excised for histological examination.
Cell separation and culture
Three patients diagnosed with NICH were enrolled in the study.
Specimens of these individuals were collected through surgical
treatment at the Shanghai Ninth People’s Hospital, Shanghai Jiao Tong
University School of Medicine (Shanghai, China). The study was approved
by the Institutional Medical Ethical Review Board of Shanghai Ninth
People’s Hospital. Informed consent was provided for the specimens,
according to the Declaration of Helsinki. The clinical diagnosis was
confirmed by both the Department of Pathology and the Department of
Plastic and Reconstructive Surgery at Shanghai Ninth People’s Hospital.
Detailed patient information is provided in Supplementary Table S1.
Vascular endothelial cells were isolated from specimen of congenital
hemangioma. The tissue was digested with 0.2% collagenase I (Roche
Diagnostics, Indianapolis, IN).The tissue homogenate was filtered
through a 70-μm cell strainer (Fisher Scientific, Hampton, NH). Red
blood cells (RBCs) were lysed using Red Blood Cell Lysis Buffer
(C3702,Beyotime). Then, centrifuge at 300g for 10 min, discard the
supernatant. Resuspend cells in pre-chilled PBS. Resuspend cells in 100
μL buffer for every 10^7 cells. Vascular endothelial cell of congenital
hemangioma selected using anti-CD31-coated magnetic beads (Miltenyi
Biotec, Germany). CD31-selected cells were cultured in Endothelial Cell
Growth Medium-2 (C-22011, PromoCell) with 10% fetal bovine serum and 1%
antibiotics. All cells were incubated at 37 °C in a carbon dioxide
incubator set at 5%.
In vivo murine model of congenital hemangioma
Six-week-old female nude mice were obtained from Shanghai JieSiJie
Laboratory Animal Co.Ltd. The mice were anesthetized by 2% chloral
hydrate (0.004 mL/g) and an ABS rodent anesthesia machine (Yuyan
Corporation, Shanghai, China). The vascular endothelial cells were
(1 × 10^6) were suspended in 100 μL of 1:1 PBS and Matrigel TM (BD
Bioscience) and subcutaneously injected into the right flank of
6-week-old female nude mice (n = 3 per group). Mice were subjected to
MN patches treatment starting 2 weeks after cell injection. The
Matrigel implants were collected 4 weeks after MN patches treatment.
All mice were euthanized after implants collection. These transplants
were collected for H&E, immunohistochemistry (IHC) and
immunofluorescence (IF). The microvascular density was measured using a
previously described technique [[87]39], Microvascular counting was
performed within the same field at a magnification of 200× (0.74 mm^2).
Subsequently, the number of microvessels per 1 mm^2 was calculated and
referred to as "microvascular density (counts/mm^2)."For IHC and IF,
five images in the slides were randomly selected from each group and
captured under microscopy.
RNA-based next-generation sequencing (RNA-seq)
Total RNA of vascular endothelial cells of hemangiomas treated with
bleomycin or PBS 24 h was purified using RNeasy mini kit (Qiangen,
Germany). The preparation of the complementary DNA library and
sequencing followed the standard protocol provided by Illumina. For the
analysis of differential gene expression, the edgeR package was
employed. Genes with an absolute log2 fold change greater than 1 and a
q-value less than 0.05 were considered significantly modulated and
retained for further analysis. Pathway analysis was performed using
Gene Set Enrichment Analysis (v3.0). (SRP472027).
Statistical analysis
Analyses were performed by GraphPad 7.0 software. Data are presented as
mean ± standard error of the mean (S.E.M.). Unpaired two tailed
Student’s t-test and one-way ANOVA were utilized as appropriate. No
samples, mice or data points were excluded from the analyses. For all
analyses, results were considered statistically significant with
*p < 0.05, **p < 0.01, ***p < 0.001.
Results and discussion
Design and fabrication of BLM@PLA-MNs
In order to meet clinical requirements for treating hemangiomas, we
designed and developed a drug delivery system that combines rapid drug
administration with slow release. Hyaluronic acid was chosen to prepare
the microneedles due to its excellent biocompatibility and water
solubility (Fig. [88]1a). BLM is encapsulated in PLA MPs and delivered
subcutaneously via microneedles for drug release (Fig. [89]1b–e).
The schematic diagram illustrating the effect of hemangioma treatment
is shown in Fig. [90]1f. The internal environment of BLM@PLA MPs is
presented in Fig. [91]1d. After removing the patches from the mold,
they are attached to the nude mouse lesion. Following 10 min of
subcutaneous insertion, the MN substrate is removed, and the MPs are
rapidly released to the lesion along with the tips degrading.
Subsequently, BLM is slowly released topically from PLA MPs,
facilitating long-term inhibition and treatment of hemangiomas over a
28-day period (Fig. [92]1e and f). Compared with the direct
subcutaneous release of BLM, this strategy can maintain therapeutic
effects for a more extended period. Additionally, the rapid separation
of MN tips from substrates reduces the negative impact of long-term
wearing of microneedle patches on patients' daily lives.
Synthesis and characterization of PLA and BLM@PLA-MNs
Polylactic acid (PLA) exhibits excellent biocompatibility and
degradability. In comparison with HA, its degradation rate is
relatively slow, ensuring the long-term subcutaneous release of BLM.
Moreover, PLA MPs can be prepared under relatively mild conditions,
facilitating drug loading without compromising the bioactivity of BLM.
BLM@PLA was prepared using an in-situ drug encapsulation method.
TEM images revealed the morphology, particle sizes, and regular
spherical shape of PLA MPs. The shape and size of the MPs were
maintained even when BLM was encapsulated within them (Fig. [93]2a).
Chemical analysis conducted via energy dispersive X-ray spectrometry
(EDS) showed the presence of C, N, O, S, and Cl elements in both PLA
and BLM@PLA (Fig. [94]2a, Figs. S1 and S2). Compared with PLA MPs, the
N element content of BLM@PLA MPs increased dramatically (from 0.20% to
0.73%), suggesting the successful encapsulation of BLM in PLA MPs.
Additionally, S and Cl elements were not detected in PLA MPs, while
their contents in BLM@PLA were 1.61% and 0.01%, respectively, due to
the loading of BLM. It's worth noting that the C and O element content
changed to a certain degree after BLM was encapsulated (from 73.40% to
52.05% and 26.40% to 45.60%, respectively). This change can be
attributed to the distinct proportions of C and O in BLM and PLA.
Similar results were observed in the XPS spectrogram of MPs
(Fig. [95]2b). The peak at 395–405 eV corresponded to the N element.
The change in N element content was consistent with the EDS analyses,
further confirming the successful encapsulation of BLM. Subsequently,
the high-resolution (HR) spectra of N1s were analyzed by
differentiating and fitting different peaks at the binding energies of
399.4, 401.0, and 401.8 eV, attributed to nitrogen atoms on the –C–NR2
(R = C/H), nitrogen atoms of heterocyclic amines, and –N–(C=O),
respectively. Similarly, the S and Cl elements of MPs increased
significantly after loading BLM (Fig. S3).
Fig. 2.
[96]Fig. 2
[97]Open in a new tab
Characterization of PLA MPs and MN patches. a TEM images of PLA and
BLM@PLA MPs, along with EDS analysis of MPs. b XPS spectrogram and
high-resolution spectra of N1s of PLA and BLM@PLA. c Thermo-Gravimetric
Analysis of PLA and BLM@PLA MPs. d Optical photography, SEM image, and
microscopy image (inserts) of BLM@PLA-MNs. e The schematic diagram of
the assembled flexible adhesive bandage and its actual wearing
The loading efficiency of BLM was preliminarily investigated via UV–Vis
spectrogram of BLM in an aqueous solution (Fig. S4). The characteristic
peak of BLM apparently decreased after encapsulation. The thermal
stabilities of BLM and MPs were measured under a nitrogen atmosphere at
a heating rate of 10 ℃ min^−1 (Fig. [98]2c). The degradative weight
loss of BLM occurred at approximately 227.0 ℃, well beyond the
temperature range for synthesizing BLM@PLA. While the degradation
temperature of BLM@PLA MPs slightly decreased compared to PLA MPs due
to BLM decomposition, the residue rates for PLA, BLM@PLA, and BLM were
1.43%, 1.87%, and 14.76%, respectively, with a 0.43% weight loss
difference between the two MPs. Thus, the drug loading rate of BLM can
be calculated as 3.29 wt % according to the formula mentioned above.
These results above indicate the successful synthesis of BLM@PLA-MNs.
Fabrication and characterization of MN patches
The MN patches were prepared using a two-step method. Optical
photography revealed that the synthesized MN patches were colorless and
transparent (Fig. [99]2d). SEM, optical microscopy, and fluorescent
microscope images of the patches showed that all needles had a
pyramidal shape, with approximately 320 μm sides at the base, 830 μm in
height, and a tip-to-tip spacing of about 650 μm (Fig. [100]2d and
insert, Fig. S5). The surfaces of the needles and substrate were
uniform and smooth. The cross-section of the tips was proven to be
porous via SEM images. To further illustrate the clinical use of MN
patches, we assembled a flexible adhesive bandage with the cohesive
bandage and MN patches. The practical wearing diagram was exhibited in
Fig. [101]2e. The adhesive bandage was flexible and easy to tear off
from the skin surface, demonstrating its potential practical clinical
application.
The chemical structures of the fabricated MN patches were characterized
by FT-IR spectroscopy (Fig. [102]3a). The absorption peaks of all PLA
MPs at 756 cm^−1 were assigned to the methyl group of PLA. The
characteristic peaks of BLM and BLM@PLA at 3310–3350 cm^−1 were
assigned to the secondary amine of BLM. The characteristic peak at
1750–1760 cm^−1 was attributed to the C=O of PLA. Additionally, the
characteristic peak at 1150–950 cm^−1 was attributed to the
monosaccharide of HA. Moreover, the characteristic peaks at around
1650 cm^−1 were due to the C=O of BLM. These results confirmed the
successful fabrication of BLM@PLA-MNs.
Fig. 3.
[103]Fig. 3
[104]Open in a new tab
Characterization and degradation of BLM@PLA-MNs. a FT-IR spectroscopy
of NPs and MN patches. b Graphs depicting the mechanical behavior of
the patches under compression applied by a vertical force (the insert
includes schematics of the experimental setups, and the right side
shows amplified curves between 30 and 70% strain). c SEM images of MN
tips after insertion into fresh rabbit cadaver skin for 10 min. d SEM
images illustrating the degradable process of BLM@PLA in PBS
(pH = 7.4). e In vitro transdermal release assays of BLM from MN
patches. Bars represent means ± SD (n = 3 independent samples)
Mechanical strength of BLM@PLA-MNs
To investigate the influence on the mechanical properties of
introducing PLA MPs to MN tips, we compared the differences in
compressive properties between HA-MNs and BLM@PLA-MNs (Fig. [105]3b).
Although HA-MNs displayed higher compressive stress with the same
compressive strain (greater than 5 kN) than BLM@PLA-MNs, the
compressive stress of BLM@PLA-MNs could still reach almost 3 kN, which
was well beyond the range of interest for applications in transdermal
drug delivery systems. Additionally, we emphatically observed the
strain changing tendency within 0–450 N of stress. The stress–strain
curve of BLM@PLA-MNs exhibited an obvious trend variation, possibly due
to the abrupt failure of BLM@PLA-MNs loaded MN tips. Excellent
compressive stress of MN tips is a fundamental characteristic for
penetrating the skin. As previous works have reported, HA-based MN tips
possess excellent penetration capabilities [[106]40, [107]41].
Furthermore, the penetrative ability of tips was demonstrated by
optical photography of holes and traces created by the MN patches
(loaded with rhodamine B) inserted into fresh rabbit cadaver skin (Fig.
S6a). Additionally, H&E staining images of the rabbit cadaver skin
after treatment with the MN patches further indicated successful
penetration into the skin (Fig. S6b).
The degradation of patches and in vitro drug release assay of BLM@PLA-MNs
After the BLM@PLA-MN patches were inserted into the skin for 10 min,
the substrate was removed, and BLM@PLA MPs were released with HA MN
arrays dissolving. To investigate the degradation state, BLM@PLA-MNs
were applied to fresh rabbit cadaver skin to mimic a medical
application setting. SEM images revealed the gradual dissolving of tips
in 10 min (Fig. [108]3c). In addition, the morphological changes of
BLM@PLA MPs within 28 days were investigated via SEM (Fig. [109]3d).
The BLM@PLA MPs degraded gradually within 28 days in PBS and were
basically completely degraded after 28 days. Therefore, the
slow-release of BLM was visually confirmed. Besides, the internal
morphology of MPs on the 28th day revealed a microporous structure that
could load sufficient BLM.
In previous studies, PLA MPs were generally considered slow-degrading
in PBS solution (pH = 7) at room temperature [[110]42]. To assess the
actual release of BLM in PBS, the UV spectrogram and standard curve of
BLM were initially tested (Fig. [111]3e insert). The release of BLM was
monitored within 28 days (Fig. [112]3e). As expected, once the BLM was
encapsulated into PLA MPs, the release of BLM from the synthesized
BLM@PLA MPs occurred continuously within 28 days, reaching a maximum
(138.47 ppm, 20 mL PBS solution) at 28 days. Therefore, it could be
calculated that the maximum drug release for a single MN patch was
approximately 2.77 mg (138.47 mg/L × 0.02 L = 2.77 mg), which is
significantly lower than the safe dosage for intralesional injection
treatment of hemangiomas and vascular malformations in clinical
settings [[113]43, [114]44]. The initial loading amount of BLM was
calculated as 164.50 ppm.
In the first 7 days, only 27.64% of the maximum BLM release amount
(23.26% of the initial loading amount of BLM) was released from
BLM@PLA. The releasing amount of BLM from day 7 to day 21 reached
89.09 ppm (around 64.34% of the maximum). Even from day 21 to day 28
(the last week), the amount of released BLM from BLM@PLA MPs reached
11.11 ppm, accounting for 8.02% of the maximum. The continuous release
of BLM in the long-term could be attributed to the slow degradation of
PLA MPs. More importantly, several explosive release processes of BLM
were observed from day 2 to day 3, day 5 to day 10, and day 14 to day
21. We speculated that these results were related to the specific
closed microporous structure of BLM@PLA MPs internally. More micropores
were exposed to the PBS solution with the PLA degrading, leading to an
explosive release of BLM.
Biocompatibility and safety assessment of MN patches
Ensuring biosafety is crucial for advancing the use of biomedical
materials in clinical applications. Herein, the MN patches were
assessed the biotoxicity by CCK8 assay. Human dermal fibroblast cells
were cultivated alongside MN patches, and the cell viability was
determined by measuring the OD values at 490 nm (Fig. [115]4a). The
cell viability values for HA-MNs, PLA-MNs, BLM-MNs, and BLM@PLA-MNs
were 97.85 ± 0.05%, 92.47 ± 0.02%, 91.40 ± 0.01%, and 94.27 ± 0.006%,
respectively. It was worth noting that the cell viability of
BLM@PLA-MNs was higher than that of BLM-MNs, indicating that
BLM@PLA-MNs to some extent mitigated the impact of the drug on cells.
Overall, based on ISO 10993 standards, all the prepared MN patches
mentioned above can be classified as non-toxic [[116]45]. Furthermore,
the hemocompatibility of the MN patches was assessed by measuring the
hemolysis rate against sheep red blood cells (Fig. [117]4b). It can be
observed that the hemolysis rate of all MN patches on red blood cells
was around 1%, significantly lower than the standard for biomedical
materials (hemolysis rate < 5%) [[118]46]. Meanwhile, the hemolysis
rate of BLM@PLA-MNs was lower than that of BLM-MNs, indicating that PLA
had a minor impact on the biosafety of the MN patches.
Fig. 4.
[119]Fig. 4
[120]Open in a new tab
Biocompatibility and safety assessment of BLM@PLA-MNs. a Cytotoxicity,
b hemolysis rate of MN patches. c Schematic diagram of the intradermal
irritation test of MN patches. d Irritation and skin sensitization test
of MN patches. e Representative histological analyses of dorsal
injection points by H&E staining 72 h post-operation from MN patches
treated rabbit. Scale bar: 100 μm
Moreover, an irritation and skin sensitization test is essential for
the biological evaluation of medical devices. According to ISO
10993-10:2010 guidelines [[121]47], the potential for MN-induced
irritation reactions is assessed by injecting the MN patches extract
intradermally into rabbits (Fig. [122]4c). The topical changes were
recorded at 24 h, 48 h, and 72 h after the injections, revealing no
obvious erythema, edema, or necrosis around vesicles (Fig. [123]4d).
Histological analysis further documented microscopic features of local
skin samples 72 h after the injection. Based on representative
H&E-stained images, no inflammatory cells, necrotic tissue, or hematoma
were detected in the subcutaneous tissues of any experimental group
(Fig. [124]4e). In summary, the aforementioned findings suggest that
the MN patches demonstrate satisfactory biocompatibility, rendering
them suitable for practical medical use.
Inhibitory effect evaluation of MN patches on hemangioma formation in vivo
Microneedles have emerged as a promising platform for transdermal drug
delivery due to enhanced permeability and reduced pain sensation
[[125]48]. In this study, we delved into the inhibitory effects of
microneedle (MN) patches on hemangioma formation in vivo. Initially, a
murine hemangioma model was established in female immunodeficiency mice
by implanting vascular endothelial cells (VEC) (positive for CD31, Fig.
S8b) from congenital hemangioma lesions (Fig. S8a) into Matrigel
(Fig. [126]5a and b). The lumens in this model were positively stained
for human CD31 and α-SMA, and negatively for glut-1, mirroring the
expression pattern in congenital hemangioma tissues (Fig. [127]5g).
Moreover, in the H&E sections of the control and HA-MN groups, we
observed endothelial cells with a hobnailed appearance (indicated by
red arrows), a common feature in congenital hemangioma tissues,
reinforcing the fidelity of the model.
Fig. 5.
[128]Fig. 5
[129]Open in a new tab
Evaluation of the inhibitory effect of MN patches in murine congenital
hemangioma model. a Depiction of two patients of NICH (one located in
the right temporal region and the other in the right waist). b
Schematic illustration of the murine congenital hemangioma model. c
Xenografts harvested 4 weeks after MN patch treatment. Comparison of
mean weight (d), microvascular density (e), and capillary diameter (f)
of the xenografts in each group after 4 weeks of treatment. Error bars
represent mean ± SEM. n = 3 mice in each group for (c–f). g
Representative images of H&E staining, as well as CD31, glut-1 and
α-SMA levels assessed by immunofluorescence in the xenografts of each
group. (n = 3 per group), *p < 0.05, **p < 0.01, ***p < 0.001. Scale
bar: 100 μm
To evaluate the therapeutic effect of MN patches on hemangioma in vivo,
we applied HA-MNs, PLA-MNs, BLM-MNs, and BLM@PLA-MNs in separate test
groups. After a 10-min administration, the patches were removed, and
xenografts were harvested after 30 days. Significant reductions in
xenograft weight, vessel density, and capillary diameter were observed
in the BLM-MNs and BLM@PLA-MNs groups (Fig. [130]5c–g). In comparison
to BLM-MNs, BLM@PLA-MNs exhibited lower xenograft weight and
microvascular density, with no significant difference in capillary
diameter (Fig. [131]5d–f). It's noteworthy that the HA-MNs and PLA-MNs
groups showed a mild decrease in weight and vascular density
(Fig. [132]5d and e), potentially attributed to the angiogenesis
inhibitory effects of HA [[133]49]. Importantly, no pulmonary fibrosis,
a common complication of bleomycin [[134]50], was detected in the lungs
of both BLM-MNs and BLM@PLA-MNs groups. Additionally, there were no
significant differences in liver and kidney structures (necrosis,
inflammation, steatosis) between the control group and other
experimental groups (Fig. S7). These findings underscore the prolonged
inhibitory effect and biosafety of BLM@PLA-MNs for hemangiomas.
However, the long-term effects of bleomycin cannot be ignored.
Bleomycin can cause pulmonary toxicity, leading to conditions such as
pneumonitis and pulmonary fibrosis. Although we did not observe
abnormalities in the lungs, liver, and kidneys of mice during the
28-day experimental period, the long-term safety of this treatment
remains uncertain. This is a limitation of our study. Future research
should focus on the long-term metabolism of bleomycin in the body and
its long-term side effects. Additionally, adjusting the dosage by
altering the surface area of the microneedles offers a safer treatment
approach for lesions of varying sizes and for patients of different
ages. This remains a key focus for future research.
BLM@PLA-MNs inhibit congenital hemangiomas via modulating the P53 pathway
To elucidate the molecular mechanism underlying the inhibitory effects
of BLM@PLA-MNs on congenital hemangiomas, RNA-sequencing (RNA-seq) was
conducted on vascular endothelial cells of hemangiomas treated with
bleomycin (5 μg/mL, 24 h) or PBS (control). The heatmap revealed that
919 genes were significantly up-regulated, and 607 genes were
significantly down-regulated in CH treated with bleomycin compared to
the control (fold change > 2, p value < 0.01) (Fig. [135]6a and b).
Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that
multiple pathways, such as DNA replication, cell cycle, cellular
senescence, and the P53 signaling pathway were enriched in
bleomycin-treated group (Fig. [136]6c). Consistently, Gene Set
Enrichment Analysis (GSEA) also revealed downregulation of DNA
replication and the cell cycle (Fig. [137]6d). The P53 signaling
pathway, known as a tumor suppressor pathway, has been shown to have
connections between cell cycle arrest and DNA repair. Loss of P53 also
plays a direct role in formation of the vascular malformations
[[138]51]. Moreover, bleomycin can induce DNA double-strand breaks,
which, in turn, activate P53, leading to cell cycle arrest and/or DNA
damage, thereby inhibiting vascular malformations [[139]52–[140]54].
Western blot analysis confirmed that applying bleomycin to vascular
endothelial cells of CH for 24 h induced an increase in expression of
P53 (P53/β-actin ratio:1.40) (Fig. [141]6e and f). To further confirm
the bleomycin-mediated upregulation of the P53 pathway, we conducted
P53 immunohistochemical staining analysis on the xenograft in the
hemangioma model. In line with western blot results, BLM-MNs and
BLM@PLA-MNs showed higher expression of P53, while BLM@PLA-MNs
exhibited the strongest P53 expression and the smallest formation of
congenital hemangioma (Fig. [142]6g and i). These results supported
that BLM@PLA-MNs exerted an inhibitory effect on hemangioma through the
upregulation of the P53 signaling pathway.
Fig. 6.
[143]Fig. 6
[144]Open in a new tab
BLM@PLA-MNs inhibit congenital hemangiomas through the P53 pathway. a
Heatmap representing color-coded expression levels of differentially
expressed genes (DEGs) in CH-VEC and CH-VEC + BLM (3 patients with
hemangiomas). b Volcano plot shows up- and down-regulated genes in
CH-VEC and CH-VEC after bleomycin treatment (FC > 2, p < 0.05). c KEGG
pathway enrichment analysis of DEGs. d Enrichment plots of select GSEA
pathways enriched in CH-VEC treated with bleomycin versus control,
including DNA replication and cell cycle. e, f Western blot and
quantitative analysis of P53 expression in VEC of CH treated with
bleomycin at concentration of 5 µg/mL for 24 h. β-actin served as the
loading control. Representative images (g) and quantification (i) of
P53 levels assessed by IHC in the xenografts of each group. Error bars
represent mean ± SEM, n = 3 independent experiments for (f, i),
*p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 50 μm
Conclusions
In conclusion, our study presents a straightforward approach to
synthesize BLM-loaded HA-based MN patches. BLM encapsulation occurred
during the synthesis of PLA-MPs. Successful preparation of PLA MPs and
MN patches was confirmed through SEM imaging. We thoroughly
investigated the in vitro BLM release profile from PLA MPs. The safety
of MN patches was comprehensively validated through cell viability
assays, hemolysis ratios, and irritation and skin sensitization tests
on rabbits. Moreover, we established a murine hemangioma model and
demonstrated the inhibitory effects of BLM@PLA-loaded MN patches on
hemangioma formation in vivo. Through RNA-Seq, western blot, and IHC,
we uncovered that BLM@PLA-loaded MN patches inhibit hemangioma
formation by modulating the P53 pathway. This study represents the
first systematic exploration of combining PLA microspheres with
microneedles to load BLM for treating hemangiomas. Our findings
indicate that BLM@PLA-loaded MNs hold promise as a cost-effective and
efficient treatment method for hemangiomas.
Supplementary Information
[145]Supplementary Material 1.^ (36MB, docx)
Acknowledgements