Needle-Free Transdermal Delivery of Formulations with Distinct Molecular and Rheological Properties Using Roller-Mediated Plasma, Electroporation, and Iontophoresis: An Exploratory In Vivo High-Frequency Micro-Ultrasound Study

1. Introduction

The stratum corneum is the principal barrier to cutaneous transport. Its corneocyte–lipid architecture restricts passive movement of hydrophilic, charged, high-molecular-weight, viscous, and particulate materials, motivating physical approaches that transiently modify barrier function.[1,2,3]

Electroporation uses short electrical pulses to create transient aqueous pathways, whereas iontophoresis applies electrical current to support electromigration and electro-osmosis. Because the mechanisms are distinct, sequential or combined application may broaden the range of molecules that can be transported through skin.[2,3,4,5,6] Plasma exposure has also been investigated as a pretreatment that can alter epidermal permeability, although the magnitude and reversibility of the effect depend on plasma source, dose, waveform, and exposure conditions.[7,8,9]

The model formulations were chosen to span a practical physicochemical range rather than to compare their clinical efficacy. PRGF is an autologous, leukocyte-poor and erythrocyte-free platelet preparation. [10] LongevHA (Caromed Italia, Italy) is a dual-molecular-weight hyaluronic acid formulation containing 80 mg HA per 2.5 mL together with mannitol, nicotinamide adenine dinucleotide, resveratrol, and peptides.[11,12] Radiesse is a particulate CaHA suspension whose rheology and ultrasound appearance change with dilution.[13,14]

The primary objective was to assess whether the Intraject platform could generate sequential high-frequency ultrasound findings consistent with needle-free cutaneous delivery across these formulation classes. The secondary objective was to describe how dilution influenced CaHA distribution. Clinical improvement, durability, and therapeutic efficacy were intentionally outside the study scope.

2. Materials and Methods

2.1. Study Design and Participants

This uncontrolled, exploratory in vivo proof-of-concept case series included three adults with Fitzpatrick skin types III–IV. One participant was assigned to each product category. PRGF and LongevHA were assessed in predefined facial skin treatment fields, whereas the CaHA comparison was performed in adjacent dorsal-hand fields. The CaHA sites received undiluted, 1:1 diluted, or 1:2 diluted product. The study included no replicate treatment fields, random allocation, sham controls, or repeated treatment sessions.

Eligibility required adult status, intact skin at the selected site, suitability for the assigned formulation and device, and no aesthetic or energy-based procedure in the target field during the preceding six months. Standard product- and device-specific contraindications were applied. All participants provided written informed consent for treatment and publication of de-identified observations and images.

2.2. Study Formulations

Table 1. Model formulations and treatment allocation. NAD+, nicotinamide adenine dinucleotide; PRGF, plasma rich in growth factors; CaHA, calcium hydroxylapatite.

Formulation	Treatment field	Preparation	Applied volume
PRGF–Endoret	Participant 1; facial skin	Closed-system, leukocyte-poor and erythrocyte-free PRGF; activated with 20 µL CaCl2 per 1 mL PRGF	0.5 mL
LongevHA	Participant 2; facial skin	80 mg dual-molecular-weight HA per 2.5 mL (40 mg high- and 40 mg low-molecular-weight HA), with mannitol, NAD+, resveratrol, and peptides	0.5 mL
Radiesse CaHA	Participant 3; adjacent dorsal-hand fields	Undiluted; 1:1 (one volume CaHA plus one volume sterile saline); 1:2 (one volume CaHA plus two volumes sterile saline)	0.5 mL per field

Table 1. Model formulations and treatment allocation. NAD+, nicotinamide adenine dinucleotide; PRGF, plasma rich in growth factors; CaHA, calcium hydroxylapatite.

Formulation	Treatment field	Preparation	Applied volume
PRGF–Endoret	Participant 1; facial skin	Closed-system, leukocyte-poor and erythrocyte-free PRGF; activated with 20 µL CaCl2 per 1 mL PRGF	0.5 mL
LongevHA	Participant 2; facial skin	80 mg dual-molecular-weight HA per 2.5 mL (40 mg high- and 40 mg low-molecular-weight HA), with mannitol, NAD+, resveratrol, and peptides	0.5 mL
Radiesse CaHA	Participant 3; adjacent dorsal-hand fields	Undiluted; 1:1 (one volume CaHA plus one volume sterile saline); 1:2 (one volume CaHA plus two volumes sterile saline)	0.5 mL per field

2.3. Device and Treatment Protocol

A multifunctional Intraject system (Biotec Italia, Dueville, Italy) was used.[17] The roller applicator measured approximately 2 cm in length and diameter and contained 10 rings comprising 60 blunt 0.25 mm microelectrodes. The electrodes contacted the surface but were not intended to mechanically penetrate the epidermis. The same roller acted as the treatment interface during plasma conditioning and the subsequent electrical delivery phase (Figure 1).

Treatment comprised three stages. First, the roller was operated at 75 kHz and 500 V and moved over the 2 cm field in three multidirectional passes for approximately 30 s. Second, 0.5 mL of the assigned formulation was distributed over the field. Third, the same roller was used at device display settings of 20 W and 6.5 V in a monopolar configuration, with a negative return electrode applied to the participant. Multidirectional passes continued for 5–10 min until no visible product remained on the surface.

The device was described operationally as providing electroporation, iontophoretic electromigration/electrophoresis, and electro-osmosis. Current density, delivered charge, impedance, pulse waveform, and skin-surface temperature were not independently recorded; therefore, the displayed settings cannot be directly equated with conventional constant-current iontophoresis.

2.4. High-Frequency Micro-Ultrasound

Images were acquired with the FUJIFILM VisualSonics Vevo 3100 platform and MX400 38 MHz transducer.[16] Imaging was performed at baseline, immediately after PlasmaPeel conditioning and before formulation application, immediately after completion of the delivery protocol, and, where available, 30 min after treatment. Acquisition was confined to the predefined treatment fields.

2.5. Outcome Assessment

The primary exploratory outcome was a new or altered ultrasound pattern within the dermal or superficial subcutaneous compartment after visible surface clearance of the formulation, compared with baseline and post-PlasmaPeel images. Secondary outcomes were the qualitative distribution, homogeneity, and persistence of the post-treatment pattern and the comparative appearance of the three CaHA preparations.

A physician blinded to the formulation labels qualitatively reviewed the de-identified images. The analysis did not include predefined regions of interest, pixel-based echogenicity measurement, penetration-depth calculations, tracer assays, histology, chemical recovery, or inter-rater reliability. Hydration was interpreted cautiously because dermal water may increase low-echogenic pixels, while gain, probe pressure, coupling medium, edema, and acquisition angle can independently alter brightness.[19,20]

2.6. Safety and Statistical Analysis

Pain and visible immediate reactions were assessed during treatment and through the 30 min observation period. No longer-term safety assessment was performed. Because each condition was represented by one participant or one adjacent treatment field without replication, no inferential statistics were performed. Results are descriptive.

3. Results

3.1. Procedure Feasibility and Superficial Epidermal Appearance

All participants completed the assigned sequence. After the roller-mediated delivery phase, no residual formulation was visibly present on the treated surface. Relative to baseline, images acquired after PlasmaPeel demonstrated changes in the thickness, definition, or organization of the superficial parallel echoes while continuity of the epidermal entry echo was preserved. These findings were compatible with treatment-associated modification of the superficial interface but did not demonstrate stratum-corneum ablation.

3.2. LongevHA

At baseline, the superficial interface was relatively regular, and the underlying tissue showed a dispersed fibrillar echotexture. After PlasmaPeel, the entry echo appeared mildly reorganized. Following LongevHA delivery, the dermal and superficial subcutaneous compartments demonstrated a denser, more heterogeneous fibrillar pattern with altered interface definition (Figure 2). No discrete anechoic bolus was identified. The appearance was compatible with diffuse tissue distribution or altered hydration, but ultrasound could not directly identify HA or other molecular constituents.

3.3. PRGF

The PRGF baseline image showed a continuous superficial entry echo and a moderately heterogeneous fibrillar dermal pattern. PlasmaPeel preserved continuity but altered the arrangement of the superficial parallel echoes. Immediately after PRGF delivery, the superficial dermis appeared more heterogeneous, with increased low-echogenic spaces between reflective fibrillar structures. At 30 min, the low-echogenic pattern was more diffuse and less focally irregular (Figure 3). This temporal evolution was compatible with redistribution of water-rich material, but the images did not chemically identify PRGF.

3.4. Calcium Hydroxylapatite

Pretreatment and post-PlasmaPeel images did not show a convincing coarse particulate CaHA pattern. After undiluted CaHA application, discrete intensely hyperechoic foci with localized posterior attenuation were observed, compatible with concentrated superficial deposits but not with broad diffuse distribution. The 1:1 dilution produced multiple hyperechoic foci distributed across a broader portion of the superficial subcutaneous plane, with less consistent shadowing. The 1:2 dilution produced the broadest and least sharply circumscribed change, with more diffuse integration into the surrounding echotexture (Figure 4).

The apparent gradient was therefore one of acoustic distribution rather than simple signal intensity: undiluted CaHA was focal and concentrated; the 1:1 preparation was intermediate; and the 1:2 preparation was the most dispersed. Because natural fibrous septa can also appear hyperechoic, only new patterns relative to pretreatment images were considered compatible with CaHA. The absence of matched imaging planes and quantitative analysis precluded estimates of delivered concentration or volume.

3.5. Comparative Findings and Immediate Safety

Table 2. Qualitative ultrasound findings. Interpretations describe imaging compatibility and do not establish chemical identity or penetration depth.

Condition	Superficial interface	Dermal/subcutaneous pattern	Interpretation
PlasmaPeel	Continuity preserved; parallel echoes reorganized	No formulation-specific deposit	Treatment-associated surface change; not proof of stratum-corneum ablation
LongevHA	Continuous	Denser, heterogeneous fibrillar echotexture	Compatible with diffuse distribution or altered hydration
PRGF, immediate	Continuous	Greater heterogeneity and low-echogenic spaces	Compatible with acute distribution of water-rich material
PRGF, 30 min	Continuous	More diffuse low-echogenic pattern	Compatible with redistribution/persistent hydration
CaHA, undiluted	Continuous	Focal hyperechoic deposits with attenuation	Concentrated, limited distribution
CaHA, 1:1	Continuous	Broader heterogeneous hyperechoic pattern	Intermediate distribution
CaHA, 1:2	Continuous	Broad, diffuse, less circumscribed pattern	Greatest qualitative dispersion

No participant reported pain during the procedure. No burn, bruising, clinically significant erythema, or other immediate adverse event was observed during the 30 min follow-up.

Table 2. Qualitative ultrasound findings. Interpretations describe imaging compatibility and do not establish chemical identity or penetration depth.

Condition	Superficial interface	Dermal/subcutaneous pattern	Interpretation
PlasmaPeel	Continuity preserved; parallel echoes reorganized	No formulation-specific deposit	Treatment-associated surface change; not proof of stratum-corneum ablation
LongevHA	Continuous	Denser, heterogeneous fibrillar echotexture	Compatible with diffuse distribution or altered hydration
PRGF, immediate	Continuous	Greater heterogeneity and low-echogenic spaces	Compatible with acute distribution of water-rich material
PRGF, 30 min	Continuous	More diffuse low-echogenic pattern	Compatible with redistribution/persistent hydration
CaHA, undiluted	Continuous	Focal hyperechoic deposits with attenuation	Concentrated, limited distribution
CaHA, 1:1	Continuous	Broader heterogeneous hyperechoic pattern	Intermediate distribution
CaHA, 1:2	Continuous	Broad, diffuse, less circumscribed pattern	Greatest qualitative dispersion

No participant reported pain during the procedure. No burn, bruising, clinically significant erythema, or other immediate adverse event was observed during the 30 min follow-up.

4. Discussion

4.1. Principal Findings

This exploratory study examined delivery feasibility rather than therapeutic efficacy. Across three model formulation classes, disappearance of visible surface material was followed by new ultrasound patterns in the dermal or superficial subcutaneous compartments. The findings best fit diffuse delivery for PRGF and LongevHA, while CaHA behavior depended on dilution. The results therefore support formulation properties as determinants of transport through a multimodal needle-free system.

The use of plasma peel demonstrated a temporary change in the stratum corneum as a preparatory step for the diffusion of the tested substances mediated by iontophoresis and electroporation. Naturally, even if the diagnostic test used shows the structural change, perhaps a study performed with a device that provides clearer results were needed. It remains evident, however, that after 30 minutes in all treated cases, the superficial dermal area returned to its original state without any residual damage..

4.2. Influence of Formulation Properties

PRGF is predominantly aqueous and produced an immediate increase in superficial low-echogenic spaces followed by a more diffuse pattern at 30 min. LongevHA is more viscous and hydrophilic and produced a denser, heterogeneous tissue pattern without a focal bolus. These differences are consistent with formulation-dependent acoustic behavior, although ultrasound cannot distinguish product from transient edema or other water-related changes.

The CaHA comparison was particularly informative. Undiluted product produced focal, intensely reflective deposits, whereas dilution broadened the distribution pattern and reduced the prominence of discrete deposits and shadowing. This is consistent with known changes in CaHA rheology and sonographic appearance with dilution [13,14]. The 1:2 preparation showed the broadest qualitative dispersion, supporting the hypothesis that reduced viscosity and particulate concentration expand the transportable range of the platform.

4.3. Mechanistic and Imaging Considerations

Electroporation and iontophoresis act through complementary mechanisms, and experimental work has reported increased transport when they are combined.[2,3,4,5,6] Plasma conditioning may provide an additional surface effect.[7,8,9] Nevertheless, the present study did not measure current density, waveform, impedance, temperature, transepidermal water loss, or electrical resistance. It therefore cannot determine each modality’s relative contribution or establish a mechanistic causal pathway.

Micro-ultrasound enabled repeated non-invasive imaging over a short interval but remains a surrogate. CaHA may generate hyperechoic bands or particulate deposits with posterior acoustic shadowing, whereas water-rich changes may increase low-echogenic pixels. [13,14,19] These patterns are not chemically specific. Confirming molecular identity and quantifying transport would require fluorescent or radiolabeled surrogates, Raman or confocal imaging, tissue recovery, or another validated chemical assay.

4.4. Future Studies

A controlled split-field or split-face study should incorporate untreated and sham areas, topical-only application, plasma alone, electroporation–iontophoresis without plasma, randomized field allocation, standardized dose, multiple participants per formulation, fixed imaging settings, co-registered acquisition planes, quantitative low-echogenic-pixel and thickness analysis, transepidermal water loss or impedance measurement, and an independent molecular or tracer-based validation method. Longer follow-up is necessary to evaluate persistence and delayed adverse events.

5. Conclusions

The Intraject multimodal platform produced sequential high-frequency ultrasound changes compatible with needle-free cutaneous delivery of a biologic fluid, a dual-molecular-weight HA formulation, and CaHA preparations with different dilutions. PRGF and LongevHA generated diffuse post-treatment tissue patterns, whereas CaHA distribution increased as the formulation was diluted. PlasmaPeel altered the superficial epidermal entry echo without disrupting continuity. Because ultrasound does not chemically identify the material and the study lacked controls and quantitative validation, the results constitute preliminary feasibility evidence rather than definitive proof of transdermal transport or clinical efficacy.