PEG-Coated Large Mesoporous Silicas as Smart Platform for Protein Delivery and Their Use in a Collagen-Based Formulation for 3D Printing.

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6-8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140 °C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds.

Effects of formulation development methods on the stability of model protein pharmaceuticals embedded in solid lipid matrices.

This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.

Biomimetic Strategy To Reversibly Trigger Functionality of Catalytic Nanocompartments by the Insertion of pH-Responsive Biovalves.

We describe an innovative strategy to generate catalytic compartments with triggered functionality at the nanoscale level by combining pH-reversible biovalves and enzyme-loaded synthetic compartments. The biovalve has been engineered by the attachment of stimuli-responsive peptides to a genetically modified channel porin, enabling a reversible change of the molecular flow through the pores of the porin in response to a pH change in the local environment. The biovalve functionality triggers the reaction inside the cavity of the enzyme-loaded compartments by switching the in situ activity of the enzymes on/off based on a reversible change of the permeability of the membrane, which blocks or allows the passage of substrates and products. The complex functionality of our catalytic compartments is based on the preservation of the integrity of the compartments to protect encapsulated enzymes. An increase of the in situ activity compared to that of the free enzyme and a reversible on/off switch of the activity upon the presence of a specific stimulus is achieved. This strategy provides straightforward solutions for the development of catalytic nanocompartments efficiently producing desired molecules in a controlled, stimuli-responsive manner with high potential in areas, such as medicine, analytical chemistry, and catalysis.

Food allergens are transferred intact across the rat blood-placental barrier in vivo.

We investigated the mechanism of transplacental macromolecular transport in rats on the nineteenth day of pregnancy using tracers, transmission electron microscopy and immunohistochemistry. The blood-placental barrier of full-term rat placentas was composed of a trilaminar layer of trophoblast cells that separates the fetal capillaries from the maternal blood spaces: a layer of cytotrophoblasts lining the maternal blood space and a bilayer of syncytiotrophoblast surrounding the fetal capillaries. Horseradish peroxidase, intravenously injected into the maternal circulation, was found in the maternal blood spaces, the interspaces between the cytotrophoblasts and the syncytiotrophoblast I, many pits and small vesicles in the syncytiotrophoblast I, vesicles of the syncytiotrophoblast II, fetal connective tissue and fetal capillaries. Intravenously injected ovalbumin was detected in the maternal blood spaces, a trilaminar layer and the fetal capillaries. Neonatal Fc receptor (FcRn), a receptor for IgG, was localized at the maternal side of the blood-placental barrier. These results show that the structure of the rat blood-placental barrier is quite similar to the human blood-placental barrier, and non-specific macromolecules and food allergens may penetrate through the blood-placental barrier of the full-term placenta from the maternal to fetal circulation mediated by FcRn.

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer.

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.

Preparation and evaluation of thiomer nanoparticles via high pressure homogenization.

The aim of this study was to establish and evaluate a high pressure homogenization method for the preparation of thiomer nanoparticles. Particles were formulated by incorporation of the model protein horseradish peroxidase in chitosan-glutathione (Ch-GSH) and poly(acrylic acid)-glutathione (PAA-GSH) via co-precipitation followed by air jet milling. The resulting microparticles were suspended in distilled water using an Ultraturax and subsequently micronized by high pressure homogenization. Finally, resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity and release behaviour. The mean particle size after 30 cycles with a pressure of 1500 bar was 538 +/- 94 nm for particles consisting of Ch-GSH and 638 +/- 94 nm for particles consisting of PAA-GSH. Nanoparticles of Ch-GSH had a positive zeta-potential of +1.03 mv, whereas nanoparticles from PAA-GSH had a negative zeta potential of -6.21 mv. The maximum protein load for nanoparticles based on Ch-GSH and based on PAA-GSH was 45 +/- 2% and 37 +/- %, respectively. The release profile of nanoparticles followed a first order release kinetic. Thiolated nanoparticles prepared by a high pressure homogenization technique were shown to be stable and provide controlled drug release characteristics. The preparation method described here might be a useful tool for a more upscaled production of nanoparticulate drug delivery systems.

An efficient method for introducing macromolecules into living cells.

The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.

The jet stream microbeveler: an inexpensive way to bevel ultrafine glass micropipettes.

Ultrafine glass micropipettes can be easily beveled in a jet stream of grinding compound suspended in saline. The beveling is gradual and continuous, highly reliable, and can be accomplished with common laboratory apparatus. The beveled electrodes are comparable in performance to those prepared with expensive commercial bevelers.

Oral self-emulsifying delivery systems for systemic administration of therapeutic proteins: science fiction?

Objective: The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins through hydrophobic ion pairing. Method: Horseradish peroxidase (HRP), a model protein, was ion paired with sodium docusate to increase its hydrophobicity. The formed enzyme - surfactant complex was incorporated into SEDDS, followed by permeation studies across Caco-2 cell monolayer and freshly excised rat intestine. Results: Hydrophobic ion pairs (HIP) were formed between HRP and sodium docusate with the efficiency of 87.49 ± 1.35%. The formed complex maintained 60.97 ± 1.48% of the original enzyme activity. The ion pair was subsequently loaded into SEDDS with a payload of 0.1% (mass per cent, m/m). The obtained emulsion formed by SEDDS had a droplet size in the range from 20 to 200 nm with negative zeta potential. Permeation mechanism of the enzyme was energy-dependent and the encapsulation of the HIP complex in SEDDS enhanced the permeation of the enzyme through the Caco-2 cell monolayer and freshly excised rat intestine by 4 times and 2.5 times compared to the free enzyme, respectively. Conclusion: According to these findings, hydrophobic ion pairing followed by incorporation to SEDDS might be considered as a potential strategy for oral delivery of therapeutic proteins.

Photocrosslinked anhydride systems for long-term protein release.

Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, the potential of photocrosslinked polyanhydride (PA) networks as an encapsulation matrix for long-term delivery of macromolecules was studied. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) was evaluated from crosslinked networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), and poly(ethylene glycol) diacrylate (PEGDA), supplemented with calcium carbonate. Prior to incorporation into the networks, proteins were formulated by dilution in a cyclodextrin excipient followed by gelatin-based wet granulation. Protein release was quantified by activity assay (HRP) or fluorescence (FITC-BSA). Each protein was readily released from the networks with a unique release behavior. Most importantly, release of protein with retention of activity was achieved for durations ranging from 1 week to 4 months. The released HRP was additionally visualized using SDS-PAGE. In general, a more hydrophobic network resulted in slower rates of protein release. Incorporation of PEGDA into the matrices was critical for maintenance of integrity during degradation. These results suggest that this system may be useful as an injectable delivery system for long-term delivery of macromolecules.

Modulation of protein release from photocrosslinked networks by gelatin microparticles.

Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, photocrosslinked polyanhydride (PA) networks were modified by incorporation of microparticles to modulate long-term delivery of macromolecules. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) were evaluated from networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), poly(ethylene glycol) diacrylate (PEGDA), and calcium carbonate (CaCO3), supplemented with gelatin microparticles or sodium chloride crystals. Prior to incorporation into the networks, proteins were formulated into granules by dilution with a cyclodextrin excipient and gelatin-based wet-granulation. Protein release was modulated by incorporation of microparticles into photocrosslinked PA networks, presumably by enabling aqueous channels through the matrix. Furthermore, a dual release system has been demonstrated by incorporation of protein in both the PA matrix and the gelatin microparticles. These results suggest that microparticle incorporation into the photocrosslinked PA system may be a useful strategy to modulate protein release in injectable delivery systems for the long-term delivery of macromolecules. These composites present an interesting class of materials for bone regeneration applications.

Photo-crosslinkable, injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage.

Millions of patients worldwide suffer from cartilage injury and age/disease-related cartilage degeneration. However, cartilage, such as articular cartilage, is poor at self-regeneration. Current treatments are often invasive with limited efficacy. Developing minimal invasive strategies for effective cartilage repair is highly desired. Here, we report an injectable, photo-crosslinkable sericin hydrogel as a biomimetic extracellular matrix for minimal invasively repairing cartilage. Sericin was functionalized to be sericin methacryloyl (SerMA), which formed an in situ hydrogel upon UV light irradiation via photo-crosslinking. Possessing excellent biocompatibility, SerMA hydrogels were adhesive to chondrocytes, and promoted the proliferation of attached chondrocytes even in a nutrition-lacking condition. SerMA hydrogels exhibited photoluminescent property allowing real-time monitoring hydrogels' status. The mechanical properties and degradation rates (73% for SMH-1, 47% for SMH-2 and 37% for SMH-3 after 45 days) of SerMA hydrogels were readily tunable by varying methacryloyl modification degrees to meet various repair requirements. Notably, the in vivo implantation of chondrocyte-laden SerMA hydrogels effectively formed artificial cartilages after 8 weeks. Most importantly, the artificial cartilages molecularly resembled native cartilage as evidenced by high accumulation of cartilage-specific ECM components and upregulated expression of cartilage-critical genes. Together, this sericin hydrogel is a promising tissue engineering scaffold for generating artificial cartilage in vivo towards effective, minimal invasive cartilage repair.

Beyond the Roles in Biomimetic Chemistry: An Insight into the Intrinsic Catalytic Activity of an Enzyme for Tumor-Selective Phototheranostics.

Protein-assisted biomimetic synthesis has been an emerging offshoot of nanofabrication in recent years owing to its features of green chemistry, facile process, and ease of multi-integration. As a result, many proteins have been used for biomimetic synthesis of varying kinds of nanostructures. Although the efforts on exploring new proteins and investigating their roles in biomimetic chemistry are increasing, the most essential intrinsic properties of proteins are largely neglected. Herein we report a frequently used enzyme (horseradish peroxidase, HRP) to demonstrate the possibility of enzymatic activity retaining after accomplishing the roles in biomimetic synthesis of ultrasmall gadolinium (Gd) nanodots and stowing its substrate 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), denoted as Gd@HRPABTS. It was found that ca. 70% of the enzymatic activity of HRP was preserved. The associated changes of protein structure with chemical treatments were studied by spectroscopic analysis. Leveraging on the highly retained catalytic activity, Gd@HRPABTS exerts strong catalytic oxidation of peroxidase substrate ABTS into photoactive counterparts in the presence of intrinsic H2O2 inside the tumor, therefore enabling tumor-selective catalytic photoacoustic (PA) imaging and photothermal therapy (PTT). In addition, the MR moiety of Gd@HRPABTS provides guidance for PTT and further diagrams that Gd@HRPABTS is clearable from the body via kidneys. Preliminary toxicity studies show no observed adverse effects by administration of them. This study demonstrates beyond the well-known roles in biomimetic chemistry that HRP can also preserve its enzymatic activity for tumor catalytic theranostics.

Silk microspheres for encapsulation and controlled release.

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load protein drugs in active form for controlled release. The lipid was subsequently removed by methanol or sodium chloride treatments, resulting in silk microspheres consisting of beta-sheet structure and about 2 mum in diameter. NaCl treated microspheres had smoother surfaces compared to the methanol treatments based on SEM analysis, and both types of microspheres had a mixture of multilamellar and unilamellar structures. A model protein drug, horseradish peroxidase, was encapsulated in the microspheres. Freeze-thaw cycles during preparation led to higher loading of the peroxidase due to improved mixing between the silk and drug, while without this process the drug and silk remained in separate layers or domains in microspheres. This partitioning was determined with fluorescein-labeled silk and rhodamine-labeled dextran. Small molecules such as the enzyme substrate 3,3',5,5'-tetramethylbenzidine, Mw=240 Da, and its oxidized product freely diffused through the MeOH- and NaCl-processed silk microspheres so that enzyme loading and activity could be determined. Enzyme activity was retained during processing and in the final microspheres. The enzyme release profile depended on the NaCl-process used in microsphere preparation. The physically cross-linked beta-sheet structure of silk fibroin and the residual lipids in the microspheres played important roles in controlling enzyme release profiles. The silk microspheres have the potential for diverse applications where controlled protein release from biocompatible, mechanically tough, and slowly biodegradable carriers is desirable.

Horseradish and radish peroxidases eaten with fish could help explain observed associations between fish consumption and protection from age-related dementia.

A juxtaposition of regional cuisines and recent prospective studies of fish consumption in China and Japan points to fresh horseradish and/or radish (HRR) as possible contributors to delaying age-related dementia. The hypothesis is that the inverse association found sometimes between fish intake and cognitive decline is partially due to exposure of the oral cavity to active peroxidases from HRR served in conjunction with fish. This hypothesis can be tested by specifically looking at whether HRR is consumed with fish and whether such HRR is prepared in a way that preserves activity of HRR peroxidases. It is possible that by putting active HRR peroxidases in their mouths, elderly people supplement their age-diminished salivary antioxidant capacity and break down additional hydrogen peroxide (H2O2) in the oral cavity before it can migrate into the brain, thus decreasing the incidence of brain cell death induction by chronically-elevated H2O2. Intentional exposure of the oral cavity to active HRR peroxidases could be a prophylactic for delaying dementia. Because vegetable peroxidases are inactivated by gastric juices, it will be difficult to obtain benefit from HRR peroxidases' antioxidant effect via ingestion in encapsulated dietary supplements.

Design of Block Copolymer Micellar Aggregates for Co-Delivery of Enzyme and Anticancer Prodrug.

Traditional enzyme-prodrug therapy (EPT) is a two-step strategy, which has many serious deficiencies, so having a one-step EPT treatment becomes a problem of immediate interest. This study aims to achieve an effective co-delivery of horseradish peroxidase (HRP) as a kind of enzyme for prodrug activation and ethyl 3-indoleacetate (EIA) as anticancer prodrug. A ternary block copolymer PEG-PAsp(AED)-CA consisting of poly(ethylene glycol) (PEG), reduction-sensitive poly (N-(2,2'-dithiobis(ethylamine)) aspartamide) PAsp(AED), and cholic acid (CA) was synthesized and assembled into spherical micelles which encapsulated EIA in its hydrophobic core and HRP in a reduction-sensitive interlayer. TEM photographs show that the polymer micelle is around 40 nm, and the cell survival rate test shows that the EIA/HRP polymer micelle is highly lethal to human lung adenocarcinoma cells. Thus, co-delivery of EIA and HRP demonstrates great potential in cancer therapy, offering a structurally simple and highly tunable platform for the synchronous delivery of enzymes and prodrugs in EPT.

Effect of steroids on increased permeability of blood vessels of the stria vascularis after auditory ossicle vibration by a drill in otologic surgery.

OBJECTIVES: The vibration caused by drills used for middle ear surgery is considered one of the causes of postoperative sensorineural deafness. Seki et al reported that when drill-induced damage was created in the auditory ossicles of guinea pigs, permeability across the capillary vessels in the stria vascularis increased significantly with the duration of drill-induced vibration. The present study was undertaken to examine changes in permeability across the stria vascularis capillaries following vibration in experimental animals pretreated with steroids, with the goal of developing a method of preventing a vibration-induced increase in permeability across these capillaries. METHODS: After an intravenous dose of hydrocortisone and horseradish peroxidase, the auditory ossicles of guinea pigs were vibrated with a drill for 60 seconds. RESULTS: Intravenous steroid administration before vibration reduced the leakage of horseradish peroxidase from the stria vascularis capillaries after vibration. CONCLUSIONS: The findings suggested that steroids suppress the increase in permeability across the stria vascularis capillaries that results from drill-induced vibration.

Cytochemical study on uptake of exogenous peroxidase by Vx2 carcinoma cells transplanted into the rabbit.

When horse-radish peroxidase (HPO) was administered iv to Vx2 carcinoma-bearing rabbits, an HPO reaction product was detected in the lumina of blood vessels and the extracellular spaces between tumor cells in the first few minutes after administration. HPO was also seen in vesicles in tumor cells. Fifteen minutes to 1 hour after administration, the HPO reaction product was found mainly in the large membrane-bound vacuoles. Within 6--12 hours, the HPO activity gradually diminished in large membrane-bound vacuoles (lysosomes). In conclusion, exogenous HPO was rapidly incorporated into Vx2 carcinoma cells by pinocytosis, and then pinocytotic vesicles were fused with lysosomes.

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.

Feedback from visual cortical area 7 to areas 17 and 18 in cats: How neural web is woven during feedback.

To investigate the feedback effect from area 7 to areas 17 and 18, intrinsic signal optical imaging combined with pharmacological, morphological methods and functional magnetic resonance imaging (fMRI) was employed. A spatial frequency-dependent decrease in response amplitude of orientation maps was observed in areas 17 and 18 when area 7 was inactivated by a local injection of GABA, or by a lesion induced by liquid nitrogen freezing. The pattern of orientation maps of areas 17 and 18 after the inactivation of area 7, if they were not totally blurred, paralleled the normal one. In morphological experiments, after one point at the shallow layers within the center of the cat's orientation column of area 17 was injected electrophoretically with HRP (horseradish peroxidase), three sequential patches in layers 1, 2 and 3 of area 7 were observed. Employing fMRI it was found that area 7 feedbacks mainly to areas 17 and 18 on ipsilateral hemisphere. Therefore, our conclusions are: (1) feedback from area 7 to areas 17 and 18 is spatial frequency modulated; (2) feedback from area 7 to areas 17 and 18 occurs mainly ipsilaterally; (3) histological feedback pattern from area 7 to area 17 is weblike.