Comparison between R2'-based and R2*-based χ-separation methods: A clinical evaluation in individuals with multiple sclerosis.

Susceptibility source separation, or χ-separation, estimates diamagnetic (χdia) and paramagnetic susceptibility (χpara) signals in the brain using local field and R2' (= R2* - R2) maps. Recently proposed R2*-based χ-separation methods allow for χ-separation using only multi-echo gradient echo (ME-GRE) data, eliminating the need for additional data acquisition for R2 mapping. Although this approach reduces scan time and enhances clinical utility, the impact of missing R2 information remains a subject of exploration. In this study, we evaluate the viability of two previously proposed R2*-based χ-separation methods as alternatives to their R2'-based counterparts: model-based R2*-χ-separation versus χ-separation and deep learning-based χ-sepnet-R2* versus χ-sepnet-R2'. Their performances are assessed in individuals with multiple sclerosis (MS), comparing them with their corresponding R2'-based counterparts (i.e., R2*-χ-separation vs. χ-separation and χ-sepnet-R2* vs. χ-sepnet-R2'). The evaluations encompass qualitative visual assessments by experienced neuroradiologists and quantitative analyses, including region of interest analyses and linear regression analyses. Qualitatively, R2*-χ-separation tends to report higher χpara and χdia values compared with χ-separation, leading to less distinct lesion contrasts, while χ-sepnet-R2* closely aligns with χ-sepnet-R2'. Quantitative analysis reveals a robust correlation between both R2*-based methods and their R2'-based counterparts (r ≥ 0.88). Specifically, in the whole-brain voxels, χ-sepnet-R2* exhibits higher correlation and better linearity than R2*-χ-separation (χdia/χpara from R2*-χ-separation: r = 0.88/0.90, slope = 0.79/0.86; χdia/χpara from χ-sepnet-R2*: r = 0.90/0.92, slope = 0.99/0.97). In MS lesions, both R2*-based methods display comparable correlation and linearity (χdia/χpara from R2*-χ-separation: r = 0.90/0.91, slope = 0.98/0.91; χdia/χpara from χ-sepnet-R2*: r = 0.88/0.88, slope = 0.91/0.95). Notably, χ-sepnet-R2* demonstrates negligible offsets, whereas R2*-χ-separation exhibits relatively large offsets (0.02 ppm in the whole brain and 0.01 ppm in the MS lesions), potentially indicating the false presence of myelin or iron in MS lesions. Overall, both R2*-based χ-separation methods demonstrated their viability as alternatives to their R2'-based counterparts. χ-sepnet-R2* showed better alignment with its R2'-based counterpart with minimal susceptibility offsets, compared with R2*-χ-separation that reported higher χpara and χdia values compared with R2'-based χ-separation.

χ-Separation Imaging for Diagnosis of Multiple Sclerosis versus Neuromyelitis Optica Spectrum Disorder.

Background Use of χ-separation imaging can provide surrogates for iron and myelin that relate closely to abnormal changes in multiple sclerosis (MS) lesions. Purpose To evaluate the appearances of MS and neuromyelitis optica spectrum disorder (NMOSD) brain lesions on χ-separation maps and explore their diagnostic value in differentiating the two diseases in comparison with previously reported diagnostic criteria. Materials and Methods This prospective study included individuals with MS or NMOSD who underwent χ-separation imaging from October 2017 to October 2020. Positive (χpos) and negative (χneg) susceptibility were estimated separately by using local frequency shifts and calculating R2' (R2' = R2* - R2). R2 mapping was performed with a machine learning approach. For each lesion, presence of the central vein sign (CVS) and paramagnetic rim sign (PRS) and signal characteristics on χneg and χpos maps were assessed and compared. For each participant, the proportion of lesions with CVS, PRS, and hypodiamagnetism was calculated. Diagnostic performances were assessed using receiver operating characteristic (ROC) curve analysis. Results A total of 32 participants with MS (mean age, 34 years ± 10 [SD]; 25 women, seven men) and 15 with NMOSD (mean age, 52 years ± 17; 14 women, one man) were evaluated, with a total of 611 MS and 225 NMOSD brain lesions. On the χneg maps, 80.2% (490 of 611) of MS lesions were categorized as hypodiamagnetic versus 13.8% (31 of 225) of NMOSD lesions (P < .001). Lesion appearances on the χpos maps showed no evidence of a difference between the two diseases. In per-participant analysis, participants with MS showed a higher proportion of hypodiamagnetic lesions (83%; IQR, 72-93) than those with NMOSD (6%; IQR, 0-14; P < .001). The proportion of hypodiamagnetic lesions achieved excellent diagnostic performance (area under the ROC curve, 0.96; 95% CI: 0.91, 1.00). Conclusion On χ-separation maps, multiple sclerosis (MS) lesions tend to be hypodiamagnetic, which can serve as an important hallmark to differentiate MS from neuromyelitis optica spectrum disorder. © RSNA, 2022 Supplemental material is available for this article.

Brighter spotty lesions on spinal MRI help differentiate AQP4 antibody-positive NMOSD from MOGAD.

In a large acute myelitis cohort, we aimed to determine whether brighter spotty lesions (BSLs)-using the refined terminology-on spinal magnetic resonance imaging (MRI) help distinguish aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) from myelin oligodendrocyte glycoprotein antibody disease (MOGAD). An experienced neuro-radiologist and two neurologists independently analyzed 133 spinal MRI scans (65 from MOGAD and 68 from AQP4-NMOSD) acquired within 1 month of attacks. BSLs were observed in 18 of 61 (30%) participants with AQP4-NMOSD, while none of 49 participants with MOGAD showed BSL (p < 0.001). BSL during the acute phase would be useful to differentiate AQP4-NMOSD from MOGAD.

χ-separation: Magnetic susceptibility source separation toward iron and myelin mapping in the brain.

Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method, χ-separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

Intracellular Uptake Mechanism of Bioorthogonally Conjugated Nanoparticles on Metabolically Engineered Mesenchymal Stem Cells.

Nanoparticles have been used for effectively delivering imaging agents and therapeutic drugs into stem cells. However, nanoparticles are not sufficiently internalized into stem cells; thus, new delivery method of nanoparticles into stem cells is urgently needed. Herein, we develop bicyclo[6.1.0]nonyne (BCN)-conjugated gold nanoparticles (BCN-AuNPs), which can be bioorthogonally conjugated to azide (-N3) groups on the surface of metabolically engineered stem cells via bioorthogonal click chemistry. For incorporating azide groups on the cell surface, first, human adipose-derived mesenchymal stem cells (hMSCs) were metabolically engineered with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz). Second, clickable BCN-AuNPs were bioorthogonally conjugated to azide groups on Ac4ManNAz-treated hMSCs. Importantly, a large amount of BCN-AuNPs was specifically conjugated to metabolically engineered hMSCs and then internalized rapidly into stem cells through membrane turnover mechanism, compared to the conventional nanoparticle-derived endocytosis mechanism. Furthermore, BCN-AuNPs entrapped in endosomal/lysosomal compartment could escape efficiently to the cytoplasm of metabolically engineered stem cells. Finally, BCN-AuNPs in stem cells were very safe, and they did not affect stem cell functions, such as self-renewal and differentiation capacity. These bioorthogonally conjugated nanoparticles on metabolically engineered stem cells can enhance the cellular uptake of nanoparticles via bioorthogonal conjugation mechanism.

Recurrence of clinical events at the same anatomical location in patients with MOG antibody-associated disease.

OBJECTIVES: Likelihood of clinical events occurring within the same anatomical location in patients with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) was retrospectively investigated. METHODS: A total of 236 clinical events in 90 patients with MOGAD from nine referral hospitals were analyzed via logistic regression, and odds ratios (ORs) were calculated. Anatomical lesion location was divided into four groups; optic nerve, spinal cord, cerebral hemisphere, and brainstem/cerebellum. RESULTS: At all locations, there was an increased likelihood of a second attack occurring at the same location as the initial event (cerebral hemisphere OR = 22.14, brainstem/cerebellum OR = 18.4, spinal cord OR = 9.1, and optic nerve OR = 7.8). There was an increased likelihood of a third attack occurring at the same location as the initial event in the optic nerve (OR = 14.9), cerebral hemisphere (OR = 11.7), and spinal cord (OR = 6.7). There were positive trends toward a third clinical event occurring at the same location as the first and/or second events if the event was in the optic nerve (OR = 13.5), cerebral hemisphere (OR = 6.9), or spinal cord (OR = 5.7). CONCLUSIONS: The current study suggests that clinical relapses of MOGAD during early stage tend to recur at the same anatomical locations in the central nervous system.

Comparison of MOG and AQP4 antibody seroprevalence in Korean adults with inflammatory demyelinating CNS diseases.

We aimed to compare seroprevalence of anti-myelin oligodendrocyte glycoprotein (MOG) and anti-aquaporin-4 (AQP4) antibodies in Korean adults with inflammatory demyelinating diseases (IDDs) of the central nervous system (CNS), based on a multicenter nationwide database. Sera were analyzed using a live cell-based assay for MOG and AQP4 antibodies. Of 586 Korean adults with IDDs of the CNS, 36 (6.1%) and 185 (31.6%) tested positive for MOG and AQP4 antibodies, respectively. No participant showed double positivity. Seroprevalence of MOG antibodies was about five times lower than that of AQP4 antibodies in a large cohort of Korean adults with IDDs of the CNS.

Recurrent Transient Neurological Deficit Due to Intracerebral Steal Phenomenon in Association with a Developmental Venous Anomaly.

We report a symptomatic developmental venous anomaly (DVA) not causing parenchymal abnormality to provide a pathophysiologic clue in patients with recurrent transient neurologic deficit. A 32-year-old male presented with recurrent transient motor aphasia and headache in the left fronto-temporal region for three years. The symptoms usually lasted for one hour. Brain computed tomography (CT) angiography and magnetic resonance imaging using gradient recalled echo showed a prominent penetrating vein at the left frontal periventricular region. Brain CT perfusion imaging performed during the symptoms revealed increased perfusion in the corresponding area with relatively decreased perfusion in the left fronto-temporal cortices. Digital subtraction angiography revealed collecting venous blood from the left septal and thalamostriate veins draining into the left cavernous sinus without early arteriovenous shunting. In this patient, an inciting incident might have led to imbalance of the venous flow surrounding the DVA, causing venous hypertension and the intracerebral steal phenomenon in the surrounding area. The relatively hypoperfused cortical area adjacent to the DVA could be considered the cause of the transient motor aphasia, while venous hypertension could be the cause of the headache.

Application of the 2017 McDonald diagnostic criteria for multiple sclerosis in Korean patients with clinically isolated syndrome.

OBJECTIVES: To evaluate the validity of the revised 2017 McDonald criteria for multiple sclerosis (MS) compared with the 2010 McDonald criteria to predict conversion to clinically definite multiple sclerosis (CDMS) in patients with clinically isolated syndrome (CIS). METHODS: A total of 163 patients from seven referral hospitals in Korea, who experienced a first clinical event suggestive of MS between 2006 and 2017, were enrolled. Patients were stratified into two groups according to outcome at the last visit: CDMS converters who experienced a second clinical event and non-converters. RESULTS: Of the 163 patients with a mean follow-up of 63 months, 60% converted to CDMS. The sensitivity, specificity, positive and negative predictive values and accuracy were, respectively, 88.8%, 43.1%, 70.2%, 71.8% and 70.6% for the 2017 McDonald criteria and 53.1%, 69.2%, 72.2%, 49.5% and 59.5% for the 2010 McDonald criteria. After exclusion of 82 patients who received disease-modifying agents before the second attack, the specificity of the 2017 and 2010 McDonald criteria increased to 85.0% and 95.0%, but sensitivity decreased to 83.6% and 47.5%, respectively. CONCLUSION: The 2017 McDonald criteria afforded higher sensitivity and accuracy but lower specificity compared with the 2010 McDonald criteria for prediction of conversion to CDMS in Korean CIS patients.

Evaluation of 2016 MAGNIMS MRI criteria for dissemination in space in patients with a clinically isolated syndrome.

OBJECTIVES: We compared validity of 2010 McDonald and newly proposed 2016 Magnetic Resonance Imaging in Multiple Sclerosis (MAGNIMS) criteria for dissemination in space (DIS) in predicting the conversion to clinically definite multiple sclerosis (CDMS) in patients with clinically isolated syndrome (CIS). METHODS: Between 2006 and 2016, we enrolled 170 patients who had a first clinical event suggestive of multiple sclerosis (MS) from seven referral hospitals in Korea. Patients were classified into two groups based on the main outcome at the last follow-up: CDMS converters, who experienced a second attack, and non-converters. RESULTS: Of 170 patients with mean follow-up duration of 54 months, 51% converted to CDMS. The sensitivity, specificity, accuracy, and positive and negative predictive values of 2010 McDonald criteria were 70.9%, 63.1%, 67.1%, 66.3%, and 67.9%, and those for 2016 MAGNIMS criteria were 88.4%, 46.4%, 67.7%, 62.8%, and 79.6%, respectively. When we excluded 80 patients who underwent disease-modifying therapy before the second clinical event, the specificity increased to 92.3% and 84.6%, but the sensitivity decreased to 58.8% and 82.4% for 2010 McDonald and 2016 MAGNIMS criteria, respectively. CONCLUSION: 2016 MAGNIMS magnetic resonance imaging (MRI) criteria for DIS showed higher sensitivity but lower specificity than 2010 McDonald criteria in predicting conversion to CDMS in CIS patients.

Lifespan, growth rate, and body size across latitude in marine Bivalvia, with implications for Phanerozoic evolution.

Mean body size in marine animals has increased more than 100-fold since the Cambrian, a discovery that brings to attention the key life-history parameters of lifespan and growth rate that ultimately determine size. Variation in these parameters is not well understood on the planet today, much less in deep time. Here, we present a new global database of maximum reported lifespan and shell growth coupled with body size data for 1 148 populations of marine bivalves and show that (i) lifespan increases, and growth rate decreases, with latitude, both across the group as a whole and within well-sampled species, (ii) growth rate, and hence metabolic rate, correlates inversely with lifespan, and (iii) opposing trends in lifespan and growth combined with high variance obviate any demonstrable pattern in body size with latitude. Our observations suggest that the proposed increase in metabolic activity and demonstrated increase in body size of organisms over the Phanerozoic should be accompanied by a concomitant shift towards faster growth and/or shorter lifespan in marine bivalves. This prediction, testable from the fossil record, may help to explain one of the more fundamental patterns in the evolutionary and ecological history of animal life on this planet.

Idiopathic aquaporin-4 antibody negative longitudinally extensive transverse myelitis.

BACKGROUND: Longitudinally extensive transverse myelitis (LETM) is a characteristic manifestation of neuromyelitis optica (NMO). However, not all patients with LETM are positive for aquaporin-4 (AQP4) antibodies. We evaluated the characteristics of idiopathic isolated LETM negative for AQP4 antibodies. METHODS: From the National Cancer Center registry of inflammatory diseases of the central nervous system, patients with LETM as an initial manifestation and follow-up for at least two years were enrolled. Their medical records and MRIs were reviewed retrospectively. AQP4 antibody was confirmed by three different validated methods at least three times. Cerebrospinal fluid (CSF) glial fibrillary acidic protein (GFAP) levels were measured to investigate astrocyte damage. RESULTS: Among 108 patients with first-ever LETM, 55 were positive for AQP4 antibodies (P-LETM) and 53 were consistently negative. Of them, seven were later diagnosed with seronegative NMO, and four were positive for MOG antibodies. The remaining 42 patients (N-LETM) showed several features distinct from P-LETM: male predominance, older age of onset, milder clinical presentation, spinal cord confinement and absence of combined autoimmunity. CSF GFAP levels were not increased in N-LETM but were markedly elevated in P-LETM. CONCLUSIONS: Idiopathic isolated N-LETM is not that rare among first-ever LETM, and has many features distinct from P-LETM where astrocytic damage is evident.

Cognitive correlates of cerebral vasoreactivity on transcranial Doppler in older adults.

BACKGROUND: This study was performed to explore the possible contributions of cerebral hemodynamic changes to the cognitive impairment in patients with Alzheimer's disease (AD). METHODS: A total of 194 participants were included: 52 controls, 75 patients with mild cognitive impairment (MCI), and 67 patients with AD. Demographic characteristics, vascular risks, mini-mental state examination (MMSE), and clinical dementia rating (CDR) were assessed, and magnetic resonance imaging of the brain was performed to evaluate white matter hyperintensities (WMHs). Using transcranial Doppler (TCD) ultrasonography, cerebrovascular reactivity (CVR) was evaluated with a breath-holding test, in addition to the mean blood flow velocity (MFV), pulsatility index (PI), and resistance index (RI) of the middle cerebral artery. RESULTS: After adjusting for covariates such as age, education, WMH severity, and vascular risks, TCD parameters such as MFV, PI, and RI did not differ between the 3 groups. However, CVR was significantly reduced in the AD group (45.33 ± 11.49%), compared with the other groups (56.36 ± 14.65%, controls; 53.84 ± 15.47%, MCI group; P < .001). Multiple regression analyses also showed that CVR was associated with MMSE scores. CVR differed according to the CDR scores (P < .001). CONCLUSIONS: Our finding may be suggestive of an underlying microangiopathic mechanism in AD patients. Furthermore, there was an association between the impaired function of cerebral microvessels and cognitive impairment. Further research is needed to fully establish whether altered cerebral hemodynamics may be considered an independent factor in predicting cognitive decline or an effect of pathologic processes involved in AD.

The usefulness of brain MRI at onset in the differentiation of multiple sclerosis and seropositive neuromyelitis optica spectrum disorders.

BACKGROUND: Although neuromyelitis optica (NMO) is a central nervous system (CNS) autoimmune disease distinct from multiple sclerosis (MS). NMO and NMO spectrum disorder (NMOSD) sometimes show asymptomatic lesions on brain magnetic resonance imaging (MRI) at onset, and even present with symptomatic brain involvement. OBJECTIVES: We investigated whether brain MRI at onset can be helpful for the differentiation of MS and NMOSD. METHODS: We retrospectively analyzed initial brain MRIs, performed within three months of onset, in patients with MS (n = 51) and anti-aquaporin4-antibody-positive patients with NMOSD (n = 67). RESULTS: NMOSD patients met the Paty (37%) and Barkhof (13%) criteria, and the criteria of the European Magnetic Imaging in MS (MAGNIMS) study group (9%), for MS. Ovoid lesions perpendicular to the lateral ventricle, isolated juxtacortical lesions in U-fibers and isolated ovoid/round cortical lesions were found only in MS patients, whereas longitudinal corticospinal tract lesions, extensive hemispheric lesions, periependymal lesions surrounding the lateral ventricle and cervicomedullary lesions were found only in NMOSD patients. CONCLUSIONS: Our study suggests that it is difficult to differentiate MS from NMOSD by the fulfillment of the MRI criteria for MS on brain MRI at onset; however, the characteristic morphology of brain lesions is highly useful for the early differentiation of the two disorders.

Disability weight for each level of the Expanded Disability Status Scale in multiple sclerosis.

BACKGROUND: The Expanded Disability Status Scale (EDSS) is the most widely employed ordinal disability scale in multiple sclerosis (MS). However, how far apart the individual EDSS levels are along the disability spectrum has not been formally quantified. OBJECTIVES: The objective of this paper is to generate refined disability weights (DWs) for each of the ordinal EDSS levels. METHODS: We performed the person trade-off (PTO) procedure to derive DWs of five representative EDSS categories (2, 4, 6, 7 and 9), and DWs of the remaining EDSS categories were assigned by linear interpolation. The modified Delphi process was used to achieve consensus among raters. RESULTS: DWs were 0.021 for EDSS 2, 0.199 for EDSS 4, 0.313 for EDSS 6, 0.617 for EDSS 7, and 0.926 for EDSS 9. Panel members achieved a high degree of consensus for each DW, as indicated by low coefficients of variation. CONCLUSIONS: Our DWs confirmed that EDSS is an ordinal scale with highly variable intervals. The availability of DW for each EDSS level allows direct comparison of each MS outcome state with other health states and provides a foundation for the estimation of the disability-adjusted life-years lost of individual patients.

Evaluation of McDonald MRI criteria for dissemination in space in Korean patients with clinically isolated syndromes.

We investigated the validity of the 2005 and 2010 McDonald magnetic resonance imaging (MRI) criteria for dissemination in space (DIS) to predict the conversion of clinically definite multiple sclerosis (CDMS) in 94 Korean patients with clinically isolated syndromes (CIS). The sensitivity, specificity, accuracy, positive and negative predictive values of the 2005, 2010 McDonald DIS criteria were comparable to those observed in the Caucasian population. This finding suggests that after careful exclusion of alternative explanations, particularly neuromyelitis optica (NMO) and NMO spectrum disorder (NMOSD), both the 2005 and 2010 McDonald DIS criteria are also useful to predict conversion to CDMS in Korean patients with CIS.

Diffuse cerebral vasospasm with infarct after intrathecal cytarabine in childhood leukemia.

Although the varied neurotoxicity of intrathecal (IT) chemotherapy for treatment of childhood acute leukemia is well known, most are related to transient post-puncture headache, drug-induced arachnoiditis, or leukoencephalopathy after methotrexate or cytarabine. Cerebral vasospasm leading to acute infarct after IT chemotherapy is very uncommon in children. Reported herein is a rare case of diffuse cerebral vasospasm with subsequent cerebral infarct after IT cytarabine in a 7-year-old boy with acute lymphoblastic leukemia, who successfully recovered with supportive management, and a review of the literature.

Performance of Large Language Models on the Korean Dental Licensing Examination: A Comparative Study.

PURPOSE: This study investigated the potential application of large language models (LLMs) in dental education and practice, with a focus on ChatGPT and Claude3-Opus. Using the Korean Dental Licensing Examination (KDLE) as a benchmark, we aimed to assess the capabilities of these models in the dental field. METHODS: This study evaluated three LLMs: GPT-3.5, GPT-4 (version: March 2024), and Claude3-Opus (version: March 2024). We used the KDLE questionnaire from 2019 to 2023 as inputs to the LLMs and then used the outputs from the LLMs as the corresponding answers. The total scores for individual subjects were obtained and compared. We also compared the performance of LLMs with those of individuals who underwent the exams. RESULTS: Claude3-Opus performed best among the considered LLMs, except in 2019 when ChatGPT-4 performed best. Claude3-Opus and ChatGPT-4 surpassed the cut-off scores in all the years considered; this indicated that Claude3-Opus and ChatGPT-4 passed the KDLE, whereas ChatGPT-3.5 did not. However, all LLMs considered performed worse than humans, represented here by dental students in Korea. On average, the best-performing LLM annually achieved 85.4% of human performance. CONCLUSION: Using the KDLE as a benchmark, our study demonstrates that although LLMs have not yet reached human-level performance in overall scores, both Claude3-Opus and ChatGPT-4 exceed the cut-off scores and perform exceptionally well in specific subjects. CLINICAL RELEVANCE: Our findings will aid in evaluating the feasibility of integrating LLMs into dentistry to improve the quality and availability of dental services by offering patient information that meets the basic competency standards of a dentist.

Nanonization and Deformable Behavior of Fattigated Peptide Drug in Mucoadhesive Buccal Films.

This study was tasked with the design of mucoadhesive buccal films (MBFs) containing a peptide drug, leuprolide (LEU), or its diverse nanoparticles (NPs), for enhanced membrane permeability via self-assembled nanonization and deformable behavior. An LEU-oleic acid conjugate (LOC) and its self-assembled NPs (LON) were developed. Additionally, a deformable variant of LON (d-LON) was originally developed by incorporating l-α-phosphatidylcholine into LON as an edge activator. The physicochemical properties of LON and d-LON, encompassing particle size, zeta potential, and deformability index (DI), were evaluated. MBFs containing LEU, LOC, and NPs (LON, d-LON) were prepared using the solvent casting method by varying the ratio of Eudragit RLPO and hydroxypropyl methylcellulose, with propylene glycol used as a plasticizer. The optimization of MBF formulations was based on their physicochemical properties, including in vitro residence time, dissolution, and permeability. The dissolution results demonstrated that the conjugation of oleic acid to LEU exhibited a more sustained LEU release pattern by cleaving the ester bond of the conjugate, as compared to the native LEU, with reduced variability. Moreover, the LOC and its self-assembled NPs (LON, d-LON), equivalent to 1 mg LEU doses in MBF, exhibited an amorphous state and demonstrated better permeability through the nanonization process than LEU alone, regardless of membrane types. The incorporation of lauroyl-L-carnitine into the films as a permeation enhancer synergistically augmented drug permeability. Most importantly, the d-LON-loaded buccal films showed the highest permeability, due to the deformability of NPs. Overall, MBF-containing peptide NPs and permeation enhancers have the potential to replace parenteral LEU administration by improving LEU druggability and patient compliance.