Helper Lipid-Enhanced mRNA Delivery for Treating Metabolic Dysfunction-Associated Fatty Liver Disease.

Lipid nanoparticles (LNPs) represent the forefront of mRNA delivery platforms, yet achieving precise delivery to specific cells remains a challenge. The current targeting strategies complicate the formulation and impede the regulatory approval process. Here, through a straightforward regulation of helper lipids within LNPs, we introduce an engineered LNP designed for targeted delivery of mRNA into hepatocytes for metabolic dysfunction-associated fatty liver disease (MAFLD) treatment. The optimized LNP, supplied with POPC as the helper lipid, exhibits a 2.49-fold increase in mRNA transfection efficiency in hepatocytes compared to that of FDA-approved LNPs. CTP:phosphocholine cytidylyltransferase α mRNA is selected for delivery to hepatocytes through the optimized LNP system for self-calibration of phosphatidylcholine levels to prevent lipid droplet expansion in MAFLD. This strategy effectively regulates lipid homeostasis, while demonstrating proven biosafety. Our results present a mRNA therapy for MAFLD and open a new avenue for discovering potent lipids enabling mRNA delivery to specific cells.

Detection and elimination of trace d-lactic acid in lignocellulose biorefining chain: Generation, flow, and impact on chiral lactide synthesis.

High chiral purity of lactic acid is a crucial indicator for the synthesis of chiral lactide as the primary intermediate chemical for ring-open polymerization of high molecular weight polylactic acid (PLA). Lignocellulose biomass is the most promising carbohydrate feedstock for commercial production of PLA, but the presence of trace d-lactic acid in the biorefinery chain adversely affects the synthesis and quality of chiral lactide. This study analyzed the fingerprint of trace  d-lactic acid in the biorefinery chain and found that the major source of  d-lactic acid comes from lignocellulose feedstock. The naturally occurring lactic acid bacteria and water-soluble carbohydrates in lignocellulose feedstock provide the necessary conditions for  d-lactic acid generation. Three strategies were proposed to eliminate the generation pathway of  d-lactic acid, including reduction of moisture content, conversion of water-soluble carbohydrates to furan aldehydes in pretreatment, and conversion to  l-lactic acid by inoculating engineered  l-lactic acid bacteria. The natural reduction of lactic acid content in lignocellulose feedstock during storage was observed due to the lactate oxidase-catalyzed oxidation of  l- and  d-lactic acids. This study provided an important support for the production of cellulosic  l-lactic acid with high chiral purity.

Development of gestational diabetes mellitus in women with periodontitis in early pregnancy: A population-based clinical study.

AIM: This study aimed to determine whether periodontitis in early pregnancy and periodontal therapy during gestation affect the incidence of gestational diabetes mellitus (GDM) through a population-based clinical study. MATERIALS AND METHODS: Subjects without periodontitis at 1-4 weeks of gestation who met our inclusion criteria were enrolled in the non-periodontitis group. Periodontitis patients who agreed or refused to receive periodontal therapy during pregnancy were separately enrolled in the periodontitis treated or untreated group. At 12-16 weeks of gestation, gingival crevicular fluid (GCF) and venous blood were collected for analyses of bacterial species and serum inflammatory mediators, respectively. At 24-28 weeks of gestation, GDM patients were identified by oral glucose tolerance tests. The association tests were performed using Chi-squared statistics and regression analyses. RESULTS: The complete data of 3523 pregnant women were recorded during the study period. GDM incidence among the untreated periodontitis participants (84/749, 11.21%) was significantly higher than that among the non-periodontitis participants (108/2255, 4.79%) (p < .05), and periodontal treatment during gestation reduced the incidence from 11.21% (untreated group) to 7.32% (38/519, treated group) (p < .05). Based on multiple logistic regression analyses, it was found that periodontitis in early pregnancy was associated with GDM, and three-step regression analyses showed that Porphyromonas gingivalis (P. gingivalis) and the serum TNF-α and IL-8 levels played a role in the association between untreated periodontitis and GDM. Furthermore, Pearson's correlation test indicated that the existence of P. gingivalis in GCF was positively correlated with high serum levels of these two inflammatory mediators. CONCLUSIONS: This study establishes a connection between periodontitis in early pregnancy and GDM and demonstrates that the presence of P. gingivalis is associated with high levels of inflammatory mediators in serum, and thereby may contribute to the development of GDM. In-depth mechanistic studies are needed to further support these findings.

Black Phosphorus-Based Multimodal Nanoagent: Showing Targeted Combinatory Therapeutics against Cancer Metastasis.

In breast cancer chemophotothermal therapy, it is a great challenge for the development of multifunctional nanoagents for precision targeting and the effective treatment of tumors, especially for metastasis. Herein, we successfully design and synthesize a multifunctional black phosphorus (BP)-based nanoagent, BP/DTX@PLGA, to address this challenge. In this composite nanoagent, BP quantum dots (BPQDs) are loaded into poly(lactic-co-glycolic acid) (PLGA) with additional conjugation of a chemotherapeutic agent, docetaxel (DTX). The in vivo distribution results demonstrate that BP/DTX@PLGA shows striking tropism for targeting both primary tumors and lung metastatic tumors. Moreover, BP/DTX@PLGA exhibits outstanding controllable chemophotothermal combinatory therapeutics, which dramatically improves the efficacy of photothermal tumor ablation when combined with near-light irradiation. Mechanistically, accelerated DTX release from the nanocomplex upon heating and thermal treatment per se synergistically incurs apoptosis-dependent cell death, resulting in the elimination of lung metastasis. Meanwhile, in vitro and in vivo results further confirm that BP/DTX@PLGA possesses good biocompatibility. This study provides a promising BP-based multimodal nanoagent to constrain cancer metastasis.

A magnetic mucus-penetrating nanoagent boosting phlegm elimination for inhalation injury treatment.

Inhalation injuries arising from exposure to toxic gases or smoke in fires or industrial accidents pose grave risks and significant respiratory complications. The limited efficacy of current treatment strategies stems from challenges in delivering therapeutic agents across the mucus barrier to the damaged trachea and bronchus. This research explores the reparative potential and underlying mechanisms of sputum-penetrable magnetic nanoparticles (MNPs) coated with poly(N-isopropylacrylamide) (PNIPAM), combined with polyethylene glycol (PEG), and loaded with ambroxol hydrochloride (AH) (MNPs@PNIPAM-AH@PEG) as an innovative therapeutic approach for inhalation injuries. The PNIPAM coating, a thermo-responsive polymer, aims to enhance targeted drug release under an external stimulus. The PEG component is designed to mitigate hydrophobic repulsion and electrostatic forces, facilitating nanoagent penetration of the mucus barrier-an obstacle in inhalation injury treatment. PEG's hydrophilicity, combined with the magnetically attracted NPs, enables deep penetration through the mucus layer adhering to the mucus epithelium. Thermal effects break the outer thermal shell of MNPs, accelerating drug release, resolving sputum, and reducing inflammation. The results showed improved therapeutic impact by significantly reducing inflammation, enhancing mucociliary clearance, and promoting tissue repair. Moreover, the MNPs@PNIPAM-AH@PEG NPs showed good biocompatibility and biosafety both in vitro and in vivo. This research underscores the potential of MNPs@PNIPAM-AH@PEG NPs as a novel therapeutic strategy for inhalation injuries, paving the way for innovative treatments in emergency medicine and respiratory care.

A new treatment strategy for hemifacial microsomia: Auricular reconstruction with an expanded two-flap method and simultaneous mandibular distraction osteogenesis.

BACKGROUND: Owing to the complex clinical manifestations of hemifacial microsomia (HFM), multidisciplinary cooperation is required to achieve better therapeutic effects in terms of function and aesthetics. This study aimed to explore the efficacy of the expanded two-flap auricular reconstruction combined with mandibular distraction osteogenesis in the treatment of HFM. METHODS: This surgical strategy was performed in three stages. In the first stage, the retroauricular skin was expanded with a tissue expander and a mandibular distraction device was installed. In the second stage, the traditional expanded two-flap method for auricular reconstruction was adapted, and the framework was fabricated with costal cartilage and wrapped with the expanded skin flap, retroauricular fascia flap, and full-thickness skin graft. In the final stage, the tragus and lobule were rebuilt, the concha cavity deepened, and the mandibular distraction device removed. RESULTS: From January 2014 to November 2018, 166 HFM patients underwent auricular reconstruction with the expanded two-flap method and simultaneous mandibular extension in our hospital. The median follow-up period was 9.3 months. Of the 166 patients, 154 patients and their families were satisfied with the results, and only 16 patients experienced complications. CONCLUSIONS: This three-stage technique of simultaneous auricular reconstruction and mandibular distraction osteogenesis is safe and effective in achieving facial symmetry, improving occlusal contact, shortening treatment course, and relieving patient's suffering, especially for HFM patients.

Surface porous poly-ether-ether-ketone based on three-dimensional printing for load-bearing orthopedic implant.

Poly-ether-ether-ketone (PEEK) possesses excellent biocompatibility and similar elastic modulus as bones but yet suffers from poor osseointegration. In order to balance PEEK's mechanical and osseointegration properties, a novel surface porous PEEK (SP-PEEK) is successfully fabricated by fused deposition modelling three-dimensional printing (FDM 3DP) and characterized by mechanical and osteogenesis in vitro tests. Moreover, the effects of pore diameter and pore layer number on the mechanical behaviors of SP-PEEK are investigated by theoretical model and numerical simulation. Comparison among experimental, theoretical and simulation results show good agreement. As pore diameter decreases, the equivalent strength and modulus become more sensitive to the decrease of pore layer number. In addition, the SP-PEEK exhibits the mechanical properties within the range of human trabecular bone and cortical bone, and thus can be tailored to mimic human bone by adjusting the pore diameter and pore layer number, which is benefit to mitigate stress shielding. The effects of pore diameter on the cell proliferation and osteogenic differentiation of SP-PEEK are tested by the co-culture of osteoblast precursor cells (MC3T3-E1) and SP-PEEK round discs. Results showcase that porous surface improves the osteogenesis in vitro, and the SP-PEEK group that the pore diameter is 0.6 mm exhibits optimal-performance osteogenesis in vitro.

Screening of potential biomarkers for Yin-deficiency-heat syndrome based on UHPLC-MS method and the mechanism of Zhibai Dihuang granule therapeutic effect.

BACKGROUND: Yin-deficiency-heat (YDH) syndrome is a subhealth state of the individual, mainly manifested as oral ulcers, dry mouth, constipation, and other symptoms. Zhibai Dihuang granule (ZDG), as a classic traditional Chinese medicine, is effective in treating YDH syndrome. We screened the potential biomarkers for diagnosing YDH syndrome, and explored the mechanisms of the therapeutic effect of ZDG. METHODS: Plasma samples from the Pinghe (PH, healthy control) group, the Shanghuo (SH, YDH syndrome) group, and the ZDG treated group (therapeutic group) were analyzed by using metabolomics profiling. The data were analyzed by multivariate statistical and bioinformatics analyses. RESULTS: We screened four differential metabolites such as, decanoylcarnitine, dodecanoylcarnitine, phosphatidylcholine (PC), and Aspartate (Asp) Arginine (Arg) Proline (Pro) in the SH group and the PH group. The results showed that the combination of above four metabolites could serve as a potential biomarker for the early diagnosis of YDH syndrome. The metabolites decanoylcarnitine and glucose were found to be differentially expressed in the YDH syndrome group and tended to be normalized after ZDG treatment. CONCLUSION: The increased levels of four differential metabolites (decanoylcarnitine, dodecanoylcarnitine, PC, and Asp Arg Pro) revealed that individuals with YDH syndrome may have increased energy metabolism in the body, which could lead to disorders of fatty acids ß-oxidation and immune function. The levels of two differential metabolites including decanoylcarnitine and glucose returned to normal after ZDG treatment, indicating that ZDG could treat YDH syndrome by regulating glucose metabolism and fatty acids ß-oxidation. Our study provides a new method for the diagnosis of YDH syndrome, and may provide theoretical basis for novel therapeutic strategies of YDH syndrome.

Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Coimmobilized IFN-γ plus TNF-α Biomaterials.

Using external methods to induce the death of cancer cells is recognized as one of the main strategies for cancer treatment. Research indicated that TNF-α is frequently used in tumor biotherapy while IFN-γ can directly inhibit tumor cell proliferation. In our study, TNF-α and IFN-γ were coimmobilized on polystyrene material (PSt) or Fe3O4-oleic acid nanoparticles (NPs). Then the structural change of these two proteins can be observed. Meanwhile, the expressions of both TNF-α and IFN-α increased significantly, as determined by gene microarray analysis; however, in the presence of TNF-α plus IFN-α inhibitors, TNF-α and IFN-α did not increase in HeLa cells induced by coimmobilized IFN-γ plus TNF-α. Our results indicate that such change can stimilate HeLa cells to secrete more TNF-α and IFN-α, by which the apoptosis of HeLa cells could be further induced. This study is the first report of autocrine-induced apoptosis of HeLa cells. In addition, we performed ELISA, RT-PCR, flow cytometry, and Western blot analyses, as well as a series of analytical tests at the animal level. our data also indicate that the PSt-coimmobilized IFN-γ plus TNF-α has apparent effects for cancer treatment in vivo, which is of great significance for translation into clinical medicine.

Occiput-axis crossing translaminar screw fixation technique using offset connectors: An in vitro biomechanical study.

OBJECTIVE: Fixation with the axis vertebra (C2) using pedicle screws is commonly used to treat an unstable occipitocervical junction; however, it is accompanied by a risk of vertebral artery injury. The occiput-C2 (OC2) crossing translaminar screw fixation technique may avoid this risk, but rod implantation is difficult. Offset connectors can help facilitate this construct. This study aimed to evaluate the stability of a technique for OC2 crossing translaminar screw fixation using offset connectors (C2LAM + OF) in comparison with other methods. PATIENTS AND METHODS: Six fresh-frozen human cadaveric occipital-cervical spines were tested intact under flexion, extension, lateral bending, and axial rotation. These were then made into a type II odontoid fracture model, instrumented with an occipital plate, and tested in the following modes: C2 bilateral pedicle screws (C2P), a single C2 pedicle screw and bilateral C3 lateral mass screws (C2P + C3M), C2 crossing translaminar screws (C2LAM), and C2LAM + OF. The OC2 range of motion (ROM) for each construct was obtained and compared using a repeated-measures analysis. RESULTS: The ROM of the C2LAM + OF construct was found not to be significantly different from that of the C2P and C2P + C3M fixations in every direction (p > 0.05). However, the C2LAM + OF construct was superior to the C2LAM construct in axial rotation (p < 0.05). CONCLUSIONS: OC2 crossing translaminar screw fixation using offset connectors offers similar stability to C2 pedicle screw fixation and is an effective alternative method for treating an unstable occipitocervical junction.

Auricular Reconstruction in Hemifacial Microsomia with an Expanded Two-Flap Method.

BACKGROUND: Reconstruction of external ear is important for the child/adult with craniofacial deformities to achieve balance and harmony of the face and head. The aim of this study was to investigate the clinical application of an expanded two-flap method for auricular reconstruction in hemifacial microsomia. METHODS: Between January of 2014 and November of 2015, 111 hemifacial microsomia patients with microtia underwent auricular reconstruction with an expanded two-flap method. The clinical data of these patients were reviewed retrospectively. Thirty-two patients (28.8 percent) underwent auricular reconstruction in combination with simultaneous mandibular lengthening. Microtia was treated by an expanded two-flap method, which includes three stages. In the first stage, the retroauricular skin was expanded using a kidney-shaped tissue expander. In the second stage, the costal cartilage was harvested and the framework was fabricated. The anterior surface of the framework was enveloped by the expanded skin flap. The posterior surface and the helical rim of the framework is covered by a retroauricular fascial flap and a full-thickness skin graft. In the third stage, the tragus was reconstructed, the lobule was formed, and the concha was excavated. The surgical planning and skills of auricular reconstruction-especially for hemifacial microsomia-were described and analyzed. The median duration of follow-up was 8.3 months. RESULTS: There were nine cases (8.1 percent) of complications in our study. During follow-up, 103 patients (92.8 percent) had satisfactory outcomes, seven (6.3 percent) had partially satisfactory outcomes, and one patient (0.9 percent) had an unsatisfactory outcome. CONCLUSION: Auricular reconstruction using an expanded two-flap method in hemifacial microsomia is safe and effective, with satisfying middle-term results. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

The role of STAT-6 as a key transcription regulator in HeLa cell death induced by IFN-γ/TNF-α co-immobilized on nanoparticles.

Based on the fact that the transcription of STAT-1 plus its Serine 727 and Tyrosine 701 phosphorylation is not the pre-requisite for the cell death signal transduction in the IFN-γ signaling pathway induced by co-immobilized IFN-γ/TNF-α, we investigate both in vitro and in vivo the key transcription regulators to promote the signal transduction of HeLa cells. It is found that IFN-γ R2 is the important death signal receptor in the HeLa cell death by RNA interference. Checking the expression of the whole transcription (STAT) protein family reveals that STAT-6 is highly expressed in comparison with the other STAT proteins. The gene silence of IFN-γ R2 leads to the down-regulation of STAT-6 and phosphorylation-STAT-6 (p-STAT-6) expressions. The successful gene silence of STAT-6 results in the reduction of HeLa cell programmed death and the expression of several important key factors related to programmed cell death (p53, Bcl-2, and Bax). More importantly, our in vivo experiments by injecting nanoparticle drug carriers with the co-immobilized IFN-γ/TNF-α into nude mice model confirm the high expression of STAT-6 and p-STAT-6. It is thus concluded that, in response to IFN-γ, the co-immobilized IFN-γ/TNF-α unusually promotes the activation of STAT-6 rather than STAT-1, resulting in the enhanced cell programmed death in HeLa. The present work reveals the gene-level molecular mechanism of IFN-γ/TNF-α co-immobilized on biomaterials as a potentially effective therapy against cancer cells.

Cell death in HeLa mediated by thermoplastic polyurethane with co-immobilized IFN-γ plus TNF-α.

In order to prohibit the toxicity of free IFN-γ plus TNF-α in treating human cervical cancer HeLa cells, two kinds of thermoplastic polyurethane (polyester/polyether) biomaterials with co-immobilized IFN-γ plus TNF-α on the surfaces are prepared. The programmed cell death of HeLa induced by these biomaterials is investigated. The surface modification of these biomaterials with co-immobilized IFN-γ plus TNF-α is performed by the photo-immobilization method, and the surface structures are characterized by various techniques. The cell morphology, cell mortality, cell cycle arrest, and functional status of caspases, upon the treatment by these biomaterials, are characterized. The results show that the as-prepared biomaterials have high inhibition activity against the growth of HeLa cells. The HeLa cells mediated by the two kinds of biomaterials are mainly arrested in the G(1) phase, while those cells mediated directly by free IFN-γ plus TNF-α are mainly arrested in the S phase. It is suggested that the programmed cell death mechanism induced by these two kinds of biomaterials is both caspase-dependent and caspase-independent. Our data provide the knowledge of microscopic surface structures and cell biology basis for synthesizing the thermoplastic polyurethane biomaterials with co-immobilized IFN-γ plus TNF-α, which are promising for novel therapeutics (e.g. drug cup) design for cervical cancer patients.

Cell cycle arrest and apoptosis of OVCAR-3 and MCF-7 cells induced by co-immobilized TNF-α plus IFN-γ on polystyrene and the role of p53 activation.

The aim of this study is to reveal the biological mechanism for high anti-cancer efficiency of co-immobilized TNF-α plus IFN-γ polymeric drug (co-immobilized drug) in mediating two gynecologic cancer cell lines: MCF-7 and OVCAR-3. The co-immobilized drug is prepared by mixing 10 ng/ml TNF-α plus 10 ng/ml IFN-γ which are then photo-immobilized onto cell culture polystyrene plates. The drug compositions and microstructures are characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The MCF-7 and OVCAR-3 cell cycle arrest and programmed cell death are checked by flow cytometry, and the expression of p53 is probed by immunofluorescence staining. The phosphorylation sites of the p53 regulation and the apoptosis key protein expressions of caspase 3, 8 and 9 are detected by western blot assay. Our data show that, in case of short treatment time (48 h) at low cytokine concentrations (20 ng/ml), the co-immobilized drug demonstrates visible effects in comparison with the treatment using TNF-α plus IFN-γ freely attached on the polymeric plate (free drug). It is revealed that the co-immobilized drug leads to significant cell arrest in the S phase or G(1) and G(2) phase and offer high efficiency in mediating a caspase-dependent apoptosis via p53 transcriptional regulation. Moreover, upon the treatment by the co-immobilized drug, the two gynecologic cancer cell lines show different phosphorylation sites of p53 and then different caspase-dependent apoptosis pathways. The present work sheds deep insights into the p53 regulation mechanism responsible for the high anti-cancer efficiency of the co-immobilized TNF-α plus IFN-γ polymeric drug against MCF-7 and OVCAR-3.

Pathway of programmed cell death in HeLa cells induced by polymeric anti-cancer drugs.

Synthesis of anticancer polymeric materials plus their biological applications is one of the most charming and active research areas in biological functional materials. However, the predominant mechanisms for controlling cancer cell viability are not yet clear. In this work, cell culture polymeric materials co-immobilized with death signal proteins interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) on the surface were prepared by photochemical method to develop an anticancer polymeric drug model. Various characterizations on the microstructures and compositions, including the Fourier transform infrared spectroscopy, UV absorption spectroscopy, fluorescence measurement, atomic force microscopy, and electron spectroscopy for chemical analysis, were performed. For addressing the biological applications, we investigated systematically the death pathways of HeLa cells attached onto the drug model by means of a series of cell-biology techniques. It was demonstrated that the IFN-γ plus TNF-α co-immobilized on the polymeric material surface exhibited more notable inhibitive effects than the free IFN-γ plus TNF-α, and the induced HeLa cells were mainly along apoptosis-like PCD with the translocation of EndoG from the cytoplasm to the nucleus. These findings indicate that the polymeric drugs with the co-immobilized IFN-γ plus TNF-α may offer significant potentials for therapeutic manipulation of human cervical cancer.

Death signal transduction induced by co-immobilized TNF-α plus IFN-γ and the development of polymeric anti-cancer drugs.

Based on our earlier work on the apoptosis in HeLa cells induced by TNF-α plus IFN-γ, we investigate how the co-immobilized TNF-α plus IFN-γ promotes the signal transduction of HeLa cells. It is found that the free TNF-α plus IFN-γ has much stronger capability than the co-immobilized TNF-α plus IFN-γ in binding with apoptosis signaling receptors TNFR1, which allows an argument that the co-immobilized TNF-α plus IFN-γ can modulate the death pathway of HeLa cells. Subsequently, we determine the cell membrane surface receptor with which the co-immobilized TNF-α plus IFN-γ binds, and probe the expression of death receptor which induces the apoptosis pathway upstream protein FADD and TRADD. Our results reveal that the death signal transduction, induced by the co-immobilized TNF-α plus IFN-γ, is mainly realized via the IFN-γ signaling pathway rather than the TNF-α one. In addition, the transcription of STAT1 plus its Serine 727 and Tyrosine 701 phosphorylation is not the pre-requisite for inducing the cell death signal transduction. It is thus suggested that the co-immobilized TNF-α plus IFN-γ promotes the activation of some unknown key markers in response to IFN-γ, and the binding of the co-immobilized TNF-α plus IFN-γ with some other TNF-α receptors results in enhanced programmed cell death in HeLa cells.

Controlled stacking of charged block copolymer micelles.

Using poly(acrylic acid)-b-poly(methyl acrylate)-b-polystyrene (PAA-b-PMA-b-PS) triblock copolymers or a mixture of different molecular weight PAA-b-PS diblock copolymers, stacks of polymeric micellar assemblies, such as disks and Y-shaped cylinders, were formed through intermicellar interactions. Whereas micelles hierarchically stacked together, micellar interactions within the stack defined a uniform micelle geometry and size for up to micrometers in length. The kinetic pathway dependence and stability of the stacked assemblies were studied, and possible intermicellar interactions between micelles within the stacks are proposed.

Unique toroidal morphology from composition and sequence control of triblock copolymers.

The mechanism by which the unique toroidal supramolecular assemblies were formed for triblock copolymers of acrylic acid (AA), methyl acrylate (MA), and styrene (S), PAA99-b-PMA73-b-PS66, was probed in this study by investigating the influences of the block copolymer compositions and sequences. Two triblock copolymers, PAA99-b-PMA73-b-PS66 and PAA99-b-PS76-b-PMA62, and two diblock copolymers, PAA99-b-PMA155 and PAA99-b-PS133, were studied under experimental solution-state conditions that involved a range of solvent/nonsolvent (tetrahydrofuran/water) compositions, each in the presence of 2,2'-(ethylenedioxy)bis(ethylamine). The resulting morphologies were determined by transmission electron microscopy. The failures to afford toroidal supramolecular assemblies from both diblock copolymers having comparable lengths of the total hydrophobic chain segment, either entirely PMA or entirely PS, and from the triblock copolymer having a reversed connection sequence for the hydrophobic (PMA and PS) segments demonstrate the unique self-assembly behaviors of triblock copolymers and the importance of the block copolymer sequence.