## Reboot Your Digestive System • Salt Lake Magazine

• January 23, 2021

2020 was a long year. So many of us have been struggling with added stress and anxiety- putting a lot of new pressures on our bodies. I for one felt these strains in my gut and in my digestive system. After months of dealing with an upset stomach and gut issues, I decided there was no better time to heal my body than the new year.

I researched around locally to find what the best juice cleanse would be for my body. Just Organic Juices is a local juicer that uses certified organic ingredients and a cold-press, juicing method to minimizes oxidization, providing a clean and healthy juice. I went for their Shed Cleanse. But don’t let the name fool you, while this cleanse can help you lose weight, it’s also the perfect remedy to flood your body with all the enzymes, vitamins and minerals to boost your immune system and clean your digestive system. Which is exactly what my body needed.

The cleanse is made up of 6 (16oz) juices per day of the cleanse. I decided to go with a 1 day cleanse but Just Organic Juices offers cleanses up to 5 days. (They also have multiple types of juice cleanses, crafted for individual needs.) I find it’s best to eat clean and raw 48 hours before doing a juice cleanse. This helps prepare your body for an easy transition. I followed the recommended 2-hour period to drink the juices, ensuring you’re drinking water in-between juices.

Following the sequential order provided by Just Organic Juices, I started my day off with their (1.) Giving Greens juice. Made up of organic spinach, cucumber, parsley, celery, dandelion, apple, pear, lemon and ginger, this juice was the exact thing I needed to jump-start my day and aid in balancing my bodies pH. Each juice in this cleanse is hand selected to provide everything your body needs to detox:

2. Grateful Grapefruit: Made up of grapefruit and mint, this juice helps increase metabolic rate. While the mint helps ease the stomach and aid digestion. This juice was tangy, but so delicious. The mint was really soothing on my stomach.

3. Inspired Roots: The name says it all, this juice is made up of lemon, celery, dandelion, beets and carrots. This is an earthy juice. A beautiful deep red color. Detoxifying and full of vitamins.

4. Cleansing Chloro: Green, green, green! This juice is made up of spinach, romaine, parsley, kale and green apples. Energizing, this juice was the mid-day boost I needed.

5. Wise Beets: Made up of lemon, ginger, beets and carrots, this juice is packed with all the vitamins your body needs- a great source of iron and fiber. This juice was also pretty earthy tasting, which I really enjoyed. It left me feeling revitalized.

6. Almond Delight: Obviously, being the sweet-tooth that I am, this was my favorite! The perfect end to a perfect cleanse. Basically a juice cleanse version of a dessert. I was so excited to drink this nut milk. Made up of raw almonds, filtered water, dates and vanilla bean. Creamy, just-sweet-enough and so filling! (Outside of the juice cleanse, this would be great as a vegan coffee creamer!)

After completing this juice cleanse, I felt surprisingly full. I was initially worried that I would be hungry all day or I’d go to sleep feeling hungry, but I was definitely satisfied. Each juice made me feel nourished and sustained. My digestive system feels renewed. And while it feels cliche to say, it’s as if my body has been rejuvenated.

One thing we can all work on moving into 2021 is listening to our bodies needs. Take time to take care of your body and mind, and support local while doing so.

## Irish Pharmacist reveals top tips to boost your immune system in 2021

• January 23, 2021

## As cases of COVID-19 remain high across the country, it’s more important than ever to ensure your immune system is in tip-top shape.

Lloyds Pharmacy Superintendent Pharmacist, Denis O’Driscoll, has some great tips to help everyone get their health in check for 2021.

O’Driscoll has laid out the five most important aspects of health and wellbeing, and they’re easier to achieve than you would think!

Here are his top tips:

#### Sleep

The impact that sleep can have on our mental and physical health is often sorely underrated.

We should be aiming for 7-8 hours, more for younger children.

You should be going to bed at the same time each evening and sleeping in a cool dark room.

To help the body ease into sleep, drinking a warm drink before bed or having a warm bath with some lavender oil can be really useful.

These steps help raise the body temperature, and then in turn when it drops your body is in a state which makes falling asleep easier.

#### Eat well

We have been told this from a young age, so it won’t come as any surprise, but eating a well-balanced diet is so important.

We should be aiming for 7 portions a day of fruit and vegetables, focusing on more vegetables than fruit as too many fruit sugars are not good for our systems.

Making sure that you get a range of whole grains, pulses, lean meats, and fishes that are high in omegas oils are also important elements in helping to boost your immune system.

#### Exercise

Although your usual form of exercise might be out of the question at the moment, there are plenty of alternatives to keep you active.

Anything that increases the pulse rate and helps to keep you supple and flexible.

There is a range of online classes to help keep you and the family active, no matter what your fitness level.

These classes can be great to help motivate you to stay active and also can help with loneliness and will feel like you’re part of a community.

If classes aren’t your thing, then challenging friends and family to see who gets rack up as many steps in a week or month can be a fun way to stay fit, while staying connected.

Don’t worry if you don’t have a fitness watch, most smartphones track your steps.

#### Vitamins & supplements

During these winter months, our bodies need some support to help us function to the best of our ability.

You should speak to your pharmacist, who will be best able to advise you on the vitamins and supplements to support you and your family.

The most popular requirements are usually Vitamin C, Vitamin D & Probiotics.

#### Probiotics

Did you know? The gut contains millions of living organisms, with up to 500 different species of bacteria in the human digestive tract.

The gut can contain bacteria that is either beneficial or harmful to our health.

Problems arise when there is an imbalance, with not enough good bacteria.

This can be a result of poor diet, illness, etc. Taking a probiotic can help restore the balance.

## Are Immunity Boosting Shots The Best Way To Fight Off Illness? –

• January 23, 2021

## Coronavirus News Roundup, January 16-January 22

• January 22, 2021

Here’s how you should think about your immunity to SARS-CoV-2 after you receive your first shot of a two-dose vaccine, according to a 1/14/21 BBC Futures story: “Pretend it didn’t happen.” In other words, assume you have acquired no immunity to SARS-CoV-2 after that first dose. “…Most vaccines require booster doses to work,” writes Zaria Gorvett. Your immune system’s first response to a vaccine typically activates B cells, which make antibodies, as well as T cells of various types, including memory T cells that stick around. The problems are that B cells are short-lived, and the body makes few memory T cells until “the second meeting” with a virus or other pathogen, the story states. The 2nd dose or booster is a re-exposure that increases the number of memory T cells, the number and quality of memory B cells, and the quality of antibodies to a pathogen, according to an Imperial College London immunologist quoted in the piece. Advice notwithstanding, the story also states the percent effectiveness that recipients can expect to kick in after receiving vaccines currently available in various countries.

For health care professionals, infectious disease specialist Dr. Paul Sax at Harvard Medical School has provided answers to frequently asked COVID-19 vaccine questions. Topics addressed include how the vaccines work; vaccine effectiveness, safety, availability; whether one vaccine is preferable to another for certain patients; guidance for immunocompromised patients; second-dose issues; and guidance for people with COVID-19 or a possible case. Sax’s replies are useful for all of us. In the New England Journal of Medicine (updated 1/11/21).

Advice for avoiding the new coronavirus variant, per this piece by Tara Parker-Pope at The New York Times (1/19/21), includes upgrading to two- or three-layer masks for errands and shopping, spending time indoors only with people from your own household, cutting back on grocery store visits and time spent in stores, avoiding crowds, not entering a store if you arrive and find it crowded, remaining socially distant, washing hands frequently, and not touching your face. But most of us still don’t need N95 medical masks, according to Dr. Ashish K. Jha at the Brown University School of Public Health, the story states. Office and grocery store workers might consider a KF94 masks, Jha is quoted as saying. They resemble N95s but have ear loops rather than elastic head loops.

A computer-simulation study of airflow inside passenger cars, published 1/1/21 in Science, supports advice to keep windows open, ideally all windows, to prevent spreading SARS-CoV-2 to others while riding together in a car, truck or other motor vehicle, writes Emily Anthes for The New York Times (1/16/21). Anthes, the author of a recent book on the air we breathe inside buildings, writes that the study simulated air flow in a “Toyota Prius driving at 50 miles per hour, with two occupants: a driver in the front left seat and a single passenger in the back right.” In the cold weather, it’s uncomfortable to keep all 4 vehicle windows open in a sedan, but half-way open can ventilate just as well as fully open, the study found, according to the story. Another alternative for the two-person ride-sharing or taxi-riding configuration: fresh air flows in a way that creates “a barrier between the driver and the passenger” if each opens the window opposite them rather than the one next to them, the research suggests, Anthes writes.

It seems that the research on the transmissibility and other pandemic effects of new variants of SARS-CoV-2 remains scant. An Oxford University epidemiologist quoted in a 1/15/21 story by Kai Kupferschmidt in Science says that a new variant called P1 and identified in Manaus, Brazil, “might have nothing to do with the new surge in infections [there]; people’s immunity might simply be waning.” And a World Health Organization physician and epidemiologist is described as saying that changes in human behavior remain the driving force behind the pandemic’s resurgence. “Even if [a] variant plays a crucial role it might be driving the boost because it is transmitted more easily, like B.1.1.7, not because it can evade the immune response,” Kupferschmidt writes. He writes later in the piece, “So far, the virus does not appear to have become resistant to Covid-19 vaccines,” according to a WHO vaccinologist.

RIP Sharon Begley, one of the best science and medical journalists of our time.

You might enjoy, “Waiting for To-Go,” by Sam Lipsyte, for the New Yorker (1/18/21).

## What is the immune system? How your body fights infection

• January 22, 2021
• The immune system is a complex network of cells, tissues, and organs that fight off infection.
• Your immune system can be affected by sleep, nutrition, hormones, and exercise.
• You can help improve your immune system, but some people are chronically immunocompromised.
• Visit Insider’s Health Reference library for more advice.

Your immune system protects your body against invaders like harmful germs, including viruses, bacteria, and fungi, and toxins from harmful chemicals.

This system comprises a large network of cells, molecules, tissues, and organs that work together via several biological processes to identify and neutralize threats. Some of the major components of your immune system include your skin, tonsils, spleen, bone marrow, white blood cells, and lymph nodes.

Several factors, including sleep, nutrition, stress, hormones, and exercise, can affect your immunity for better or for worse. Certain health conditions, medical treatments, and factors like age, pregnancy, and smoking can also affect your immunity.

Here’s what you need to know about your immune system and how it functions.

The immune system is your body’s internal defense system. According to the Cleveland Clinic, your immune system primarily does two things:

1. It protects you against germs and tries to keep you from getting sick.
2. If you do get sick, it fights the infection to help you recover.

These are some of the major components of your immune system:

• Skin: Your skin is the first line of defense against germs or toxins. It acts like a barrier that prevents germs from entering your body. It also secretes fluids and other substances that destroy germs.
• Mucous membranes: Your respiratory, reproductive, urinary, and digestive tracts are lined with mucus, which traps germs and expels them. For example, when you have a cold, your body produces mucus to flush germs out of your nose, sinuses, and airways.
• White blood cells: These cells are a key part of your immune system. They locate, identify, and attack germs. There are several types of white blood cells; each one has a specific role to play in your immune response.
• Lymph nodes: These are small glands located all over your body, including in your armpit, neck, and groin regions. They are part of your lymphatic system, which filters out germs via a clear, watery fluid known as lymph. This process can help prevent germs from reaching other parts of your body.
• Spleen: Your spleen produces, stores, and dispatches white blood cells. For instance, if your body detects germs in your bloodstream, your spleen and lymph nodes manufacture and dispatch thousands of white blood cells to fight them.
• Tonsils: Located in your throat, your tonsils trap harmful germs as soon as they enter your body, fighting them to prevent respiratory infections like influenza (flu) and pneumonia
• Bone marrow: Your bone marrow produces several different types of immune cells, including white blood cells. Millions of new blood cells are produced in your bone marrow and added to your bloodstream every day.
• Thymus: Located behind your breastbone, this small organ produces white blood cells that catalogue germs and remember them in future, so that it’s easier for your body to fight those germs if they attack you again.

## How does your immune system fight infection?

If germs get past your defenses, your body launches an immune response to fight them. This involves several types of white blood cells

• B lymphocytes: These cells produce antibodies, which are special proteins designed to identify and weaken specific germs.
• T lymphocytes: These cells attack the germs identified by the antibodies. They also release cytokines, which are small proteins that orchestrate your entire immune response by controlling the growth and activity of all your blood cells and immune cells.
• Phagocytes: These cells engulf germs and destroy them.

Once your body has dealt with a certain invader, it remembers it. If the same germ attacks you again, your body recognizes it and sends out the appropriate antibodies to fight it. This can help prevent reinfection. This ability to protect against a certain illness is known as “immunity.”

“The generation of long-lasting protective memory is one of the most unique and important characteristics of the immune system. Memory is essential in allowing individuals to defend themselves from infections to which they have previously been exposed,” says Tulip Jhaveri, MD, who specializes in pathology and infectious diseases.

Vaccines help the body develop immunity to germs and infections. This is called immunization and it counts on your immune system’s memory. Though different vaccines take different approaches to the immunization process, they all trigger your immune response to fight off a particular kind of illness (like measles, smallpox, etc.).

After you receive a vaccine, your body remembers how to fight that particular germ and generates a long-lasting immune response. Certain vaccines require a booster shot, which jogs your immune system’s memory.

## How do you check your immune system?

Blood tests that measure your levels of blood cells, immune system cells, and immunoglobulins (infection-fighting proteins) can help determine whether your immune system is healthy or not.

Jhaveri says that recurring illnesses could indicate that one’s immune system isn’t functioning as it should. Some signs of a weakened immune system include:

• Frequent or prolonged colds
• Recurring gastrointestinal problems like gas, constipation, or diarrhea
• Slow healing of wounds
• Frequent infections, like pneumonia, ear infections, or sinusitis
• Persistent fatigue
• High stress levels

Certain factors and health conditions can compromise your immunity and affect your body’s ability to fight diseases. These include:

• HIV and AIDS, which damage immune cells, making your body vulnerable to other infections
• Asthma, which causes your immune system to severely overreact to harmless substances
• Leukemia and lymphoma, which affect the normal functioning of your lymphocytes (white blood cells)
• Cancer treatments, like radiation and chemotherapy, which can also damage and destroy immune cells in the process of destroying cancer cells
• Bone marrow or organ transplants, which necessitate deliberately weakening the patient’s immune system so it doesn’t reject the donated cells
• Smoking, which harms your immune system and raises your chances of developing an illness

Age also plays a role in immunity. As you get older, your immune system is more susceptible to infections, diseases, and cancers; this is referred to as “immune senescence.” Infants and young children are also more prone to infections; their immunity develops over time as they encounter germs and their body learns how to fight them. “Even pregnancy is a state of compromised immunity to a certain extent,” says Jhaveri.

Sometimes, the immune system is unable to differentiate between harmful substances and healthy tissues, causing it to attack and destroy healthy cells, which is known as autoimmune disorder. There are over 80 types of autoimmune disorders — some of the more common ones include Type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, and celiac disease.

## Insider’s takeaway

Your immune system consists of several components working together to fight off infection-causing germs. One of the most remarkable characteristics of the immune system is that it is able to remember how to defeat germs it has encountered before; a process by which you develop immunity to that illness. Vaccination, also known as immunization, relies on your immune system’s memory to help build immunity against harmful germs that cause illnesses.

Certain health conditions and medical treatments, as well as factors like age, smoking, and pregnancy can compromise your immune system and its ability to function. Frequent illnesses and chronic fatigue are some of the signs of a weakened system. Blood tests can help determine whether your immune system is functioning as it should.

Jhaveri says your best chance at boosting your immune system is to eat a healthy diet, get plenty of sleep, exercise regularly, stop smoking, limit your alcohol consumption, and reduce stress.

## Health Addiction’s Functional Supplements Provide Perfect Harmony Between Body and Mind for Health and Wellbeing

• January 22, 2021

PALM BEACH, FL, Jan. 22, 2021 (GLOBE NEWSWIRE) — The modern approach to medicine treats symptoms.

However, if you are tired of the dog chasing its tail, functional medicine, which targets the underlying causes for illnesses, is an approach you should consider.

A leading functional nutritionist, Nathaly Marcus, has developed a holistic health philosophy along with high-quality dietary supplements.

“Our health philosophy is simple. Get people addicted to healthy lifestyle choices,” said Marcus, founder and nutritionist for Health Addiction, a wellness company in Mexico City. “At Health Addiction, we have developed functional dietary supplements that promote health, not mask symptoms.”

“Health Addiction’s research and development team developed supplements that address aging challenges, the immune system, weight, joint care, and gut health,” Ms. Marcus said. “You have to treat the entire person if you want to improve their quality of life.”

Marcus emphasized that health issues are interconnected.

“You can take pain killers to mask joint pain,” she said. “But if you are obese, the stress on your joints will still exist even if you take pain killers or drugs to strengthen your bones.”

Marcus said people need to embrace a holistic approach to their health, which is why Health Addiction developed functional supplements that address the immune system, obesity, joint problems, and gut health.

Marcus is no newcomer to functional health.

Marcus, Mexico’s first functional nutritionist, trains other nutritionists, coaches and doctors about functional medicine’s benefits.

After successfully helping people in Mexico get healthy, Health Addiction plans to introduce eight popular functional supplements to American consumers:

• ESSENTIAL 5 addresses the five most important health pillars: nervous system, gut health, immune system, and cardiovascular system. It also provides an energy boost.
• GLOW PACK helps regenerate and build healthy, radiant skin, hair and nails.
• PRE + PROBIOTIC COMPLETE FORMULA helps regenerate the gut system and support the immune system.
• THERMO BURN MAX is a unique fat burner formula for weight loss.
• GUT BALANCE optimizes gut function, decreases gut inflammation, and improves nutrient absorption.
• SPORT COLLAGEN BOOSTER promotes ligament and joint elasticity and structure.
• SPIRULINA + CHLORELLA + MORINGA COMPLEX helps detoxify the body, support the immune system and promote cell regeneration.
• GASTRO 360 optimizes proper gut function and aids in heartburn, colitis, nausea and, acid reflux problems.

“We are beginning 2021. It’s a new year. Now is the time to take stock of your health and adopt healthy lifestyle choices,” Marcus said. “Get enough sleep, eat healthy foods, exercise, and, just like more than 70 percent of American adults, find dietary supplements that will provide you with a 360-degree approach to your health. Try Health Addiction supplements.”

Robert Grant
(561) 421-3045
pr@nutrapr.com


## More vaccines could be coming soon and they could be a big boost to the rollout

• January 22, 2021

States may be scrambling for vaccines now, but the United States could see additional Covid-19 vaccines by the end of March, if not sooner.

Millions of vaccine doses could be added to the pipeline in the coming months, perhaps easing the squeeze that Americans have felt as vaccines slowly start rolling out.

Janssen, the vaccine arm of Johnson & Johnson, plans to have results from its late-stage clinical trials ready for the US Food and Drug Administration to consider within weeks.

“Johnson & Johnson is right around the corner … right around the corner means that they’re probably a couple of weeks away from getting the data looked at, to have the FDA evaluate whether or not we’re in a situation where we could move ahead and start thinking about getting it out into the public,” Dr. Anthony Fauci, the director of the National Institute of Allergy and Infectious Diseases, told CNN’s Chris Cuomo on Thursday.

Dozens of vaccines are being tested in labs around the world. At least 11 are in late stage clinical trials, according to Bio, a global trade association representing biotech companies.

In the US, there are three frontrunners.

### Johnson & Johnson

Johnson & Johnson is the most likely to nest seek emergency use authorization from the FDA.

Many experts say J&J’s Covid-19 vaccine has several advantages. While J&J is running a separate trial to test its vaccine using two doses, as Moderna’s and Pfizer’s do, the current trial uses just one dose.

“This advantage goes up in neon,” said Dr. William Schaffner, an internist and infectious disease specialist with Vanderbilt University’s Department of Health Policy. “This would be the first one to be given in a single dose.”

A single does would mean more people could be vaccinated, as none would need to be set aside to give someone a second shot. That would be a huge advantage at a time when vaccines are hard to come by.

J&J’s other advantage is that it can be stored at regular refrigerator temperatures, unlike the Pfizer vaccine, which needs special deep freezers.

“If they’re successful, these vaccines would especially be popular in the developing world, because they would be easy to store and administer,” said Dr. Rafi Ahmed, the director of the Vaccine Center at Emory University.

Schaffner said adding a vaccine like this would “really accelerate” vaccination efforts in the US too.

“In other words, we could bring the vaccine to the people,” Schaffner said, “rather than bringing the people to the vaccine.”

Johnson & Johnson’s Covid-19 vaccine was made through a collaboration of J&J’s Belgium-based vaccine division, Janssen Pharmaceutical and Beth Israel Deaconess Medical Center. It is what is known as a non-replicating viral vector vaccine, using a common cold virus called adenovirus 26.

Scientists made this vaccine by taking a small amount of genetic material that codes for a piece of the novel coronavirus and integrated it with a weakened version of adenovirus 26. J&J scientists altered this adenovirus so it can enter cells, but it doesn’t replicate and make people sick. AstraZeneca uses a similar platform, but its adenovirus comes from a chimpanzee.

The adenovirus carries the genetic material from the coronavirus into human cells, tricking them into making pieces of the coronavirus spike protein – the part it uses to attach to cells. The immune system then reacts against these pieces of the coronavirus.

“So you’re not being infected with the virus that can give you Covid-19 when you get this vaccine. It just has some of the harmless Covid virus proteins on its surface,” explained Schaffner. “So essentially it’s a sheep in wolf’s clothing, and when your immune system sees it, it responds to it and creates protection against it and in the future, against the real virus that causes Covid-19.”

Dr. Paul Offit, the director of the Vaccine Education Center at Children’s Hospital of Philadelphia, said the Moderna, Pfizer and J&J Covid-19 vaccines all take a similar approach, but there is a small difference with the J&J approach.

“In the case of the Moderna and Pfizer vaccine you’re just giving the gene in a lipid nanoparticle or a fat droplet,” Offit said. “In the case of J&J you’re giving the gene in a virus that can’t reproduce itself.”

The technology used in the Covid-19 vaccine has worked with Janssen’s Ebola vaccine.

### AstraZeneca

The AstraZeneca vaccine is already being used in a few countries. It was authorized in the UK in December, and in Brazil in January, but the FDA will likely want to use US data for any emergency use authorization in the US, Moncef Slaoui, chief adviser to the Trump administration’s Operation Warp Speed, said last Tuesday.

The AstraZeneca vaccine is also a vector vaccine. Like the Johnson & Johnson vaccine, it employs an adenovirus to carry the genetic instructions for making spike protein from the coronavirus into cells to generate antibodies. AstraZeneca’s vaccine, developed with Oxford University, uses a virus that infects chimpanzees but doesn’t make people sick.

As with the Pfizer and Moderna vaccines, this Covid-19 vaccine would require two shots.

The trials so far have had some confusing results, but show it could provide up to about 70% protection. The FDA paused US trials last year after questions about adverse reactions in volunteers, but it cleared up the questions and resumed.

The AstraZeneca vaccine could potentially go up for authorization around the end of March.

### Novavax

The Novavax Phase 3 trial started in the US in December. The trial is enrolling up to 30,000 volunteers at 115 sites in the US and Mexico.

The Novavax candidate is a protein-based vaccine created out of the genetic sequence for the novel coronavirus.

The company uses virus-like nanoparticles as a base and scientists cover the base with the genetically engineered pieces of the coronavirus spike protein.

This vaccine is given in two doses, 21 days apart.

This kind of vaccine has been successfully used with other diseases like the shot for HPV and Sanofi’s flu vaccine.

Novavax said it expects to have results with its Covid-19 vaccine by early first quarter of this year. The first quarter ends March 31.

## Sustained and targeted delivery of checkpoint inhibitors by metal-organic frameworks for cancer immunotherapy

• January 22, 2021

## Abstract

The major impediments to the implementation of cancer immunotherapies are the sustained immune effect and the targeted delivery of these therapeutics, as they have life-threatening adverse effects. In this work, biomimetic metal-organic frameworks [zeolitic imidazolate frameworks (ZIFs)] are used for the controlled delivery of nivolumab (NV), a monoclonal antibody checkpoint inhibitor that was U.S. Food and Drug Administration–approved back in 2014. The sustained release behavior of NV-ZIF has shown a higher efficacy than the naked NV to activate T cells in hematological malignancies. The system was further modified by coating NV-ZIF with cancer cell membrane to enable tumor-specific targeted delivery while treating solid tumors. We envisage that such a biocompatible and biodegradable immunotherapeutic delivery system may promote the development and the translation of hybrid superstructures into smart and personalized delivery platforms.

## INTRODUCTION

Over the past decade, metal-organic frameworks (MOFs) have been actively used as intricately engineered platforms for biomedical applications (1719). Zeolitic imidazolate frameworks (ZIF-8)—a subclass of MOFs—are crystalline solids based on Zn2+ ion subunits coordinated to organic 2-methylimidazole (mIM) ligands, resulting in the formation of highly porous structures (20). ZIF-8 has recently emerged as a potential candidate for on-demand drug delivery applications due to its biocompatibility, remarkable LC, superior stability under physiological conditions (no premature drug release), pH responsiveness, and tunable drug release properties (21, 22). In comparison to other delivery vehicles, ZIFs can encapsulate different size and charge therapeutic hosts with a high LC reproducibly and deliver them on demand. Therefore, they were used in the delivery of current breakthrough proteins such as small interfering RNA (23), CRISPR-Cas9 ribonucleoprotein (24, 25), and catalytic enzymes (26, 27). Here, we developed an efficient strategy for the sustained release and high loading of the PD-1 inhibitor, nivolumab (NV), using ZIF-8 (NV-ZIF) with the capability of working on both hematological malignancies and solid tumors (Fig. 1). Targeted delivery, in the case of solid tumors, was achieved by using cell-specific membrane coating (CC), as this technique has proved viable for enhancing targeted cancer therapy (25, 28, 29).

## RESULTS

### Design and characterization of NV-ZIF

In a typical experiment for the biomimetically mineralized growth of ZIF-8, an aqueous solution containing mIM (2.5 M, 0.9 ml) and NV (1 mg·ml−1) was mixed with a separate aqueous solution of zinc (Zn) nitrate (0.5 M, 0.1 ml) at room temperature for 20 min. The solution immediately turned opaque, indicating crystal formation. Cryogenic transmission electron microscopy (cryo-TEM) and TEM micrographs clearly illustrated octahedral crystals with an average diameter ranging between 102 and 160 nm (Fig. 2A). The energy-dispersive x-ray spectroscopy elemental mapping revealed a uniform distribution of ZIF-8–associated elements, Zn, nitrogen (N), and carbon (C); and NV-associated elements, oxygen (O) and sulfur (S) (Fig. 2B).

Our powder x-ray diffraction (PXRD) results demonstrate that the embedded NV did not result in any change in ZIF-8 crystallinity (Fig. 3A), which is consistent with other reported MOF-based protein carriers (18, 26, 30). The LC and loading efficiency (LE) were then evaluated using the Bradford assay. The LC and LE were found to be 5.07 ± 1% and 80 ± 3%, respectively. NV content in NV-ZIF was also estimated by thermogravimetric analysis (TGA), and the results were comparable to those obtained by the Bradford assay (fig. S1, A and B). Comparing the thermogram of NV-ZIF with that of ZIF-8 gives information about the formulation’s composition. The TGA spectrum of the NV-ZIF showed a weight loss of 6.1% between 10° and 150°C due to the loss of the adsorbed moisture. The 23% loss observed between 250° and 444.6°C can be attributed to the pyrolysis of the carboxyl or hydroxyl groups, which most probably originated from the NV decomposition. The final range of temperature from 320° to 600°C resulted in an obvious mass loss of 52.9%, which we assigned to the removal of the organic linker molecules and the collapse of ZIF-8. The interaction between ZIF-8 and NV delayed the pyrolysis process of NV that is coved by the in situ growth of ZIF-8. Unlike NV-ZIF, ZIF-8 showed a weight loss of 3.7% between 10° and 150°C, 21.6% between 250° and 444.6°C, and 47.89% from 320° to 600°C. The 23% weight loss between 250° and 444°C supports the presence of the NV. On the basis of TGA analysis, we carried out a calcination process at 320°C for 2 hours to confirm that NV is majorly embedded at the surface of the framework, as previously reported for protein-embedded MOFs (31). The TEM image of NV-ZIF after calcination supports the existence of small cavities (fig. S1C) resulting from the removal of NV molecules and their aggregates. The ultraviolet–visible–near-infrared (IR) (UV-Vis-NIR) spectrum of NV-ZIF clearly showed the absorbance band of NV at 280 nm. The embedded NV resulted in reducing its symmetry; therefore, a red shift was observed (fig. S1D) as previously reported (32). The embedded NV was also confirmed by Fourier transform IR (FT-IR) spectrum through the absence of the vibrational band at 1660 cm−1, characteristic of COO group of NV (fig. S1E). We further investigated the possible coordination of NV and Zn2+. Therefore, different ratios of NV and Zn2+ (0.1:1 and 1:1) were stirred for 20 min at room temperature. Zn nitrate showed an absorbance band at 300 nm according to our UV spectrum (fig. S2A). When NV was mixed with Zn at different ratios, an absorbance peak at 280 nm appeared for NV only, indicating that no coordination occurred between Zn2+ and NV. The increase in the absorbance of NV is attributed to the increase in NV concentration. The FT-IR analysis showed no shift in the vibrational band at 1700 cm−1 for the COOH and 3400 cm−1 for the N─H, supporting no coordination with Zn2+ and validating the importance of mIM in the NV-ZIF formation (fig. S2B).

To monitor the loading and release, we labeled NV with rhodamine B, Rh (RNV) (fig. S3). By measuring the Rh fluorescence intensity, the majority of NV was embedded in ZIF-8 (fig. S3, A and B). Moreover, the sustained release of RNV from NV-ZIF at varying pH values in phosphate-buffered saline (PBS) was monitored by fluorescence spectroscopy (Fig. 3, B and C). NV-ZIF exhibited a slow sustained release at acidic pH (6.5), and approximately 50% of RNV was released within 12 hours. More stable release of small dosages of encapsulated RNV was observed after 24 hours, reaching more than 70% of RNV release from ZIF-8 (Fig. 3B and fig. S3D). Scanning electron microscopy images of the release process show a gradual dissociation of NV-ZIF at acidic conditions over time. NV-ZIF morphology remarkably changed after 12 hours, extrapolating the 50% release of RNV from ZIF-8 (fig. S3E). In contrast, less than 25% of RNV was released over 3 days under physiological conditions at pH 7.3, and the system exhibited an excellent colloidal stability for over 6 months (Fig. 3B and fig. S3C). Such slow and controlled release behavior is intended to improve treatment outcomes. Next, we evaluated the in vitro cytotoxicity of NV, ZIF-8, and NV-ZIF by cell counting kit-8 (CCK-8) against human embryonic kidney (HEK), HeLa, and Michigan Cancer Foundation-7 (MCF-7). As expected, no obvious cytotoxicity was observed, supporting NV-ZIF biocompatibility (fig. S4, A to C). Our system exhibited excellent NV sustained release performances and triggered pH responsiveness, which is consistent with previously reported drug-loaded ZIF-8 delivery systems (30, 32).

### Expression of PD-1 and in vitro pH-responsive NV-ZIF

The expression of PD-1 was first examined on Jurkat T cells that were either activated with anti-CD3/anti-CD28 antibodies or left unactivated. Flow cytometric analysis demonstrated that the expression of PD-1 on activated Jurkat T cells (aTCs; >80%, P < 0.005) was significantly higher than that on unactivated Jurkat T cells (10%, P < 0.005) (fig. S5, A and B). To assess NV activity and integrity after loading, we extracted NV from ZIF-8 by adding EDTA to dissociate the ZIF-8 crystals by breaking the coordination bonds between Zn2+ and 2-mIM. The released NV was then incubated with aTC for 30 min and labeled with a secondary phycoerythrin (PE)–labeled anti-human antibody. The fluorescence intensity of the extracted NV shifted to the right; the same shift was observed with free NV–treated aTC that was used as a control (fig. S5C). The same result was obtained when the residual NV in the supernatant was used, confirming that embedding NV in the framework did not affect the activity and the integrity of this antibody. Subsequently, the release of NV from NV-ZIF was tested in vitro by incubating NV-ZIF with aTC for 6 and 12 hours at slightly acidic (pH 6.5) and physiological (pH 7.3) conditions. The PD-1/anti–PD-1 (NV) interaction was detected by staining aTC against PE-labeled anti-human antibody. No obvious difference was detected when aTC was incubated with NV-ZIF for 6 hours (17.3%). Twelve hours post incubation induced the release of NV (fig. S6). A marked increase was observed at pH 6.5 (66.3%) (fig. S6B). On the other hand, treating aTC with free NV for 30 min showed a rapid binding of anti–PD-1 (NV) with PD-1 on aTC (47.7%) (fig. S6A). Such treatment profile is consistent with the one currently used in the clinics, which is associated with increased levels of toxicity (33). Administering the same dose but at a slower rate would help to avoid adverse reactions. Hence, the slow and sustained release behavior of NV-ZIF will help in reducing immune-related life-threatening events associated with free NV delivery.

### Efficacy of NV sustained release on hematological malignancies

Lymphocytes of patients with acute myeloid leukemia (AML) and chronic lymphoid leukemia (CLL) are known to express high levels of PD-1 (34, 35), while PD-L1 was shown to be up-regulated on cancerous cells and antigen-presenting cells (APCs) from these patients (3638). Peripheral blood mononuclear cells (PBMCs) isolated from patients with AML and CLL were initially treated with ZIF-8 to test its effect on T cell proliferation (CD8+ and CD4+) using Ki-67 as a marker for cell proliferation. Our data demonstrated that CD8+ and CD4+ T cells treated with phytohemagglutinin (PHA) resulted in high Ki-67 expression compared to ZIF-8 and the control (fig. S7, A to C), indicating that ZIF-8 has no effect on lymphocyte proliferation. Next, we treated CD8+ T cells with NV-ZIF for 6 and 12 hours. Compared to free NV, NV-ZIF boosted the activity of PHA-stimulated T cells over time. As shown in Figs. 4 and 5, contrary to ZIF-8, NV-ZIF enhanced the activation of CD8+ T cells compared to nontreated cells as evidenced by the higher levels of CD8+ interferon-γ (IFN-γ) and CD8+ tumor necrosis factor–α (TNF-α) T cells. NV-ZIF increased the level of CD8+ IFN-γ and CD8+ TNF-α T cells 12 hours following treatment (Figs. 4 and 5). This is mostly due to the sustained release of NV from the NV-ZIF over time. The level of activation of those cells at 12 hours was either slightly higher or comparable to cells treated with free NV (Figs. 4 and 5). Although we expected that there is higher cytokine release at 12 hours compared to 6 hours, the comparably high levels of cytokines observed for cells treated with NV-ZIF and NV suggest that there was a sufficient release of NV from the NV-ZIF.

### Cell type–specific delivery of NV

Attending to the tumor microenvironment (TME) is crucial when developing therapies for solid tumors. We modified the NV-ZIF to specifically deliver and release NV into the TME, enabling local activation of tumor-specific immune responses and reducing systemic toxicity associated with NV administration. CC membrane was used as a targeting agent in our study, which provided a personalized tumor-specific PD-1 blockade therapy. We previously validated that coating ZIF-8 with CC resulted in preferential accumulation of coated ZIF-8 within CCs from which the membrane was extracted (25). The same protocol was followed for coating NV-ZIF with MCF-7 membrane. TEM micrographs of MCF membrane–coated NV-ZIF (NV-ZIFMCF) showed an octagonal crystal with an average size of 166 nm, and the negative staining of NV-ZIFMCF exhibited rough surface after coating (fig. S8A). The PXRD patterns and intensity of NV-ZIFMCF are similar to those of the NV-ZIF and ZIF-8, which supports that the ZIF-8 crystallinity was maintained after coating (fig. S8B). Zeta potential measurements validated the complexation with CC, as the charge of the NV-ZIF dropped from +11 to −21 mV (fig. S8C). The successful functionalization was further confirmed by SDS–polyacrylamide gel electrophoresis (PAGE), followed by protein staining (fig. S9). The protein profile of the purified CC matches that of the NV-ZIFMCF (fig. S9A), indicating a good retention of the characteristic proteins inherited from the CC. Surface adhesion molecules such as CD44, E-cadherin, and CD49e were detected on CC and NV-ZIFMCF by Western blot (fig. S9B). Biocompatibility of NV-ZIFMCF was tested at different concentrations by incubation with MCF-7 for 24 hours using CCK-8. Compared to the native MCF-7, concentrations below 100 μg·ml−1 were completely safe, whereas high concentrations (100 μg·ml−1) led to a low level of cytotoxicity (fig. S10). To verify the cancer-targeting ability of NV-ZIFMCF, we incubated NV-ZIFMCF with HeLa, HEK, and MCF-7. The results indicated that NV-ZIFMCF accumulated in MCF-7 tumors and that the accumulation steadily increased with longer incubation times (fig. S11, A to C), which is consistent with our previous study (25). The targeted delivery and preferential accumulation of NV-ZIFMCF were further evaluated using homologous 4T1 cancer-bearing mice in vivo. XenoLight DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) was loaded with NV-ZIF for in vivo imaging purposes. Mice-bearing 4T1 tumors were imaged at different time points (3 and 24 hours) following injection of ZIF particles using In Vivo Imaging System (IVIS). Our data revealed that NV-ZIFMCF exhibited a high accumulation in tumors within 3 hours with prolonged tumor retention. Unlike NV-ZIFMCF, the accumulation of NV-ZIF (uncoated) was low and detected after 24 hours, supporting the efficiency of this targeting strategy (Fig. 6A). Measuring Zn2+ content of the tumor by inductively coupled plasma mass spectrometry (ICP-MS) showed a significant increase in the accumulation of NV-ZIFMCF at tumor site compared to that of NV-ZIF (Fig. 6B). Unlike most developed delivery systems that targeted superficial TME, such as melanoma (39, 40), our engineered NV-ZIFMCF efficiently targets TME inside the body. This highly specific cancer recognition ability of NV-ZIFMCF can extensively enhance the therapeutic effect of NV, as the platform showed negligible toxicity to the animals as verified by the control samples.

### Efficacy of NV sustained release on solid tumors

We injected mice with either NV-ZIFMCF, NV-ZIF, CC-NV, or NV (3 mg·kg−1 per mouse for each injection) on days 3, 6, 9, and 12. The mice were then sacrificed on day 21, and tumor sizes were measured. Results in Fig. 6 (C to F) indicate that the NV-ZIFMCF treatment significantly inhibited tumor growth compared to NV-ZIF, CC-NV, or NV treatment alone. To further test the treatment effect, we observed tumor development over 21 days after various treatments and found that the antitumor activity and survival rate were significantly extended (tumor volume maintained <200 mm3, P < 0.01) after the NV-ZIFMCF treatment (Fig. 6, C to F). The survival time was slightly extended from 42 days for untreated mice to 45 and 49 days for NV-ZIF– and NVMCF-treated mice, respectively (Fig. 6F). Free NV did not show superior tumor survival compared to NV-ZIF and NVMCF, indicating the necessity of efficient NV delivery for effective tumor inhibition. In contrast, NV-ZIFMCF significantly prolonged animal survival (Fig. 6F). Mice treated with NV-ZIFMCF also showed a significantly higher production of IFN-γ and TNF-α (P < 0.001) compared to other treated groups that showed comparable levels of production (Fig. 6G). We measured the mice body weight, and as expected, experimental and control mice did not show an obvious difference in body weight (fig. S12).

Next, we characterized CC proliferation by histological assays. CC damage was detected by hematoxylin and eosin (H&E) staining upon treatment with NV-ZIFMCF (Fig. 7A). The Ki-67 staining results revealed marked reduction in Ki-67 levels after NV-ZIFMCF treatment, resulting in significant inhibition of the proliferation of tumor cells compared to other treatments (Fig. 7B). Of a particular note, functionalizing NV with the targeting agent (CC-NV) did not result in equivalent antitumor activity to that of NV-ZIFMCF. Likewise, NV-ZIF did not exhibit antitumor activity similar to that of NV-ZIFMCF, which supports insufficient NV delivery to TME in both cases. The cancer inhibition ratio reached about 73% after treatment with NV-ZIFMCF, indicating that this strategy made TME more sensitive to immunotherapy. To gain a better understanding of the effects of each treatment regimen on lymphocytes present in the TME, we analyzed the population of FoxP3+ regulatory T cells. These FoxP3+ regulatory T cells act to suppress immune responses and, in this case, antitumor immune responses. T cells from tumor tissues were harvested and analyzed by flow cytometry to determine the percentage of regulatory T cells within the total T cell population in the tumor. The percentage of FoxP3+ cells significantly decreased in NV-ZIFMCF–treated groups compared to the control and ZIF-8, whereas NV-, CC-NV–, and NV-ZIF–treated groups showed some level of reduction (Fig. 7C), supporting the enhanced production of IFN-γ and TNF-α in groups treated with NV-ZIFMCF. These results strongly confirm the anticancer efficacy of NV-ZIFMCF enabled by the inhibition of the regulatory T cells. Collectively, the antitumor activity of CC-NV was comparable to that of NV-ZIF, indicating that sufficient delivery of NV to TME is the key for enhanced NV-ZIFMCF antitumor activity. An extrapolation of these results suggests that tumor-specific delivery of NV results in (i) enriching NV within TME, leading to local inhibition of PD-1; (ii) enhancing the sensitivity of TME to anti–PD-1 blockade therapy; and (iii) systemically activating specific antitumor immune response enabled by the local inhibition of the regulatory T cells.

## DISCUSSION

ICB therapy has shown encouraging preclinical and clinical results to treat different types of tumors. Current delivery methods, however, are not antigen specific and result in the systemic blocking of regulatory pathways, leading to systemic activation of immune cells and limiting therapeutic benefits in many patients. Consequently, there is a tremendous need to improve the safety and efficacy of such treatments. Here, we demonstrated the therapeutic potential of the sustained release and targeted delivery of NV by NV-ZIF and NV-ZIFMCF on both hematological malignancies and solid tumors, respectively.

Hematological malignancies, such as leukemia, involve continuous and systemic contact between the tumor clone and the immune system. Our NV-ZIF release behavior showed a slow release of small doses of NV over time that resulted in improving the antitumor activity with longer incubation by inducing T cell activation to a level comparable to that of free NV. This kind of release behavior is expected to reduce immune-related toxicity and increase patient compliance.

On the other hand, solid tumors are characterized by confining infiltrating lymphocytes in localized tissue. The suppressive nature of TME induces the irresponsiveness to PD-1 blockade therapy. Thus, we coated our NV-ZIF with CC to enable tumor-specific recognition, reducing off-target delivery and immune-related side effects, improving the sensitivity of TME to NV, extending the retention of NV-ZIF within tumor, and eliciting tumor-specific immunity. We used the challenging 4T1 mouse breast cancer model to demonstrate the therapeutic potential of NV-ZIFMCF. Our results showed the superior antitumor activity of NV-ZIFMCF over NV, CC-NV, and NV-ZIF. Of a particular note, functionalizing NV with the targeting agent (CC-NV) did not result in equivalent antitumor activity to that of NV-ZIFMCF. Likewise, NV-ZIF did not exhibit antitumor activity similar to that of NV-ZIFMCF, which supports insufficient NV delivery to TME in both cases. The cancer inhibition ratio reached about 73% after treatment with NV-ZIFMCF, indicating that this treatment regimen made TME more sensitive to immunotherapy. Such treatment could be followed by chemotherapy or radiotherapy to completely eradicate tumor. CC coating could be used as a promising strategy to develop a personalized tumor-specific immune response as shown in previous studies (28). Our strategy has shown that local delivery of NV-ZIFMCF leads to systemic and durable activation of antitumor immune response that has the potential to reduce the risk of metastasis. Unlike most developed delivery systems that targeted superficial TME, such as melanoma (39, 40), our developed NV-ZIFMCF was efficient in targeting TME inside the body. Our strategy shows a great clinical translation potential in patients with both hematological malignancies and solid tumors because all the materials used in this system are biocompatible, and it would be a step toward developing personalized immune therapeutics treatment plans.

In summary, we have successfully loaded NV in ZIF-8 and demonstrated the potential utility of the sustained NV release in hematological malignancies and solid tumors. The sustained release behavior of NV-ZIF has shown its efficacy in activating T cells in AML and CLL. The system was further modified to enable tumor-specific targeted delivery while treating solid tumors by coating NV-ZIF with specific CC membrane. NV-ZIFMCF displayed an enhanced antitumor activity due to the preferential accumulation and prolonged retention of NV-ZIFMCF within TME that resulted in efficient NV delivery. Collectively, this work demonstrates that tackling the sustained and targeted delivery is the way forward for the broader impact of ICB therapy in the fight against cancer.

## MATERIALS AND METHODS

### Fabrication of ZIF-8, NV-ZIF, and NV-ZIFMCF

NV-ZIF was synthesized by stirring NV (1 mg·ml−1) and 2-mIM (2.5 M, 0.9 ml) for 30 min. Zinc nitrate solution (0.5 M, 0.1 ml) was slowly added under mechanical agitation for 20 min. The resulting product was collected by centrifugation and washed three times with deionized water to remove any residues. ZIF-8 was synthesized by slowly adding zinc nitrate solution (0.5 M, 0.1 ml) to 2-mIM (2.5 M, 0.9 ml). The solution was agitated for 20 min. The supernatant of NV-ZIF was collected to calculate the LC and LE by Bradford assay. NV-ZIFMCF was fabricated by mixing 1:1 weight ratio of NV-ZIF and extracted CC membrane in deionized water. The mixture was then transferred into a syringe and successively extruded through 1.0-μm and 800.0- and 450.0-nm polycarbonate membrane. The obtained NV-ZIFMCF in solution was further purified by centrifugation to remove the free CC membrane. The zeta potential of NV-ZIF was performed using a Malvern Zetasizer Nano ZS at 25°C at pH 7.3 in aqueous solutions. PXRD measurements were performed using a Panalytical X’Pert Pro X-ray powder diffractometer using the Cu Kα radiation (40 V, 40 mA, λ = 1.54056 Å) in a θ – θ mode from 20° to 90° (2θ). TEM images were obtained using FEI Tecnai 12 microscope operating at 120 kV. For visualization by TEM, samples were prepared by dropping the solution on a copper grid 300 mesh (Electron Microscopy Sciences, LC 300-Cu). Fluorescence measurements were performed on a Cary Eclipse fluorescence spectrophotometer (Varian). The slits for excitation and emission were set at 10 nm. NV was labeled with rhodamine B (Rh) by N-hydroxysuccinimide (NHS) chemistry. Briefly, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (5 mg) and NHS (2.5 mg) were mixed with NV solution (10 mg·ml−1, 1 ml), and the mixed solution was stirred for 2 hours. Rh (63 μg) was then dissolved in dimethyl sulfoxide (100 μl), and the whole solution was stirred overnight at 4°C in the dark. The dialysis technique was used to remove unreacted EDC, NHS, and Rh.

### Preparation of CC membrane

Human breast adenocarcinoma cell (MCF-7) cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin). Cells were grown in T-175 culture flasks to full confluency and then detached and washed in PBS three times by centrifuging at 500g. Then, they were suspended in a hypotonic lysing buffer consisting of 20 mM tris-HCl (pH 7.5), 10 mM KCl, 2 mM MgCl2, and 1 EDTA-free mini protease inhibitor tablet per 10 ml of solution and disrupted using a Dounce homogenizer with a tight-fitting pestle. The entire solution was subjected to 20 passes before spinning down at 3200g for 5 min. The supernatant was saved, while the pellet was resuspended in hypotonic lysing buffer and subjected to another 20 passes and spun down again. The supernatants were pooled and centrifuged at 20,000g for 20 min, after which the pellet was discarded, and the supernatant was centrifuged again at 100,000g. The pellet containing the plasma membrane material was then washed again in 10 mM tris-HCl (pH 7.5) and 1 mM EDTA. The final pellet was collected and used as a purified CC membrane.

### CC membrane protein characterization

Protein characterization was carried out using the SDS-PAGE method. The cracked CC membrane samples were suspended in lithium dodecyl sulfate loading buffer (Invitrogen). Samples were heated to 90°C for 10 min, and 20 μl of sample was loaded into each well of a NuPAGE Novex 4 to 12% bis-tris minigel, using Mops SDS as the running buffer (Invitrogen) in an XCell SureLock Electrophoresis System based on the manufacturer’s instructions. Protein staining was accomplished using Coomassie Blue (Invitrogen) and destained in water overnight before imaging. For Western blot analysis, the protein was transferred to Protran nitrocellulose membranes (Whatman) using an XCell II Blot Module (Invitrogen) in NuPAGE transfer buffer (Invitrogen) per the manufacturer’s instructions. Membranes were probed using antibodies against CD44 (clone 515; BD Biosciences), E-cadherin (clone 36; BD Biosciences), and CD49e (BD Biosciences), followed by horseradish peroxidase–conjugated anti-mouse immunoglobulin G (Cell Signaling Technology) as the secondary antibody.

### LC and LE NV-ZIF

The LC and LE of NV in ZIF-8 nanoparticles were measured with the Bradford method. First, a standard curve of NV at 595 nm was generated. Then, LE and LC of NV in ZIF-8 were obtained by analyzing residual NV in supernatants, which was collected after washing. The LE was calculated as follows

$LC=[mass loaded drug/mass of loaded drug+NPs]×100$

$LE=[mass of drug loaded/mass of initial drug]×100$

### Release of Rh-labeled NV via pH trigger

To evaluate the release of Rh-labeled NV from ZIF-8, the fluorescence signal of Rh-labeled NV was measured by using the microplate spectrophotometer. Aliquots of hydrochloric acid were added to Rh-labeled NV-ZIF (600 μg·ml−1) in PBS to reach a pH of 6.5 at 37°C. PBS only was added to the sample of pH 7.3. The supernatant of the mixture solution was obtained through centrifugation at different time points. The fluorescence of released Rh-labeled NV was monitored by fluorescent spectroscopy (excitation/emission wavelength: 540 nm/625 nm).

### In vitro release and Jurkat activation

Anti-CD3 (mouse anti-human CD3, clone: OKT3; eBioscience) was added in a 24-well plate at a final concentration of 5 μg·ml−1 prepared in PBS (300 μl per well) and incubated for 3 hours in a 37°C incubator supplied with 5% CO2. After incubation, antibody solution was removed from each well. In the same well, 106 cells·ml−1 of Jurkat cells (acute T cell leukemia human, Jurkat, clone E6-1) were resuspended in 2 ml of RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% streptomycin (Hyclone). Anti-CD28 (purified NA/LE as described in BD mouse anti-human CD28, BD) was added at a final concentration of 1 μg·ml−1 to each well. Last, interleukin-2 was added at a final concentration of 100 U·ml−1. The cells were then incubated in a 37°C humidified incubator supplied with 5% CO2 for 3 days.

### Cell viability

CCK-8 assay was performed according to the manufacturer’s protocol. Briefly, MCF-7, HEK, and HeLa cells (5 × 103 cells per well) were seeded onto a 96-well plate. After 12 hours, the culture medium was changed, and the cells were incubated with different concentrations (100, 50, 25, 12, 6, and 3 μg·ml−1) of NV, NV-ZIF, and ZIF-8 in 200 μl of DMEM at 37°C for 24 hours. The media was then discarded, and the prepared culture medium containing 10% CCK-8 solution was added into each well, including a negative control of culture media alone. After 3 hours of incubation, the absorbance was measured at 450 nm using a microplate spectrophotometer (xMark Microplate Absorbance Spectrophotometer).

### Specific targeting studies

Flow cytometric assay was used to investigate the specific targeting ability of NV-ZIFMCF. Rh–NV–ZIFMCF was used to track the uptake of the NV-ZIFMCF. Cells were seeded in six-well plates at a density of 5 × 105 cells per single well and cultured for 12 hours in 2 ml of DMEM containing 10% FBS and 1% antibiotics (penicillin-streptomycin). After NV-ZIFMCF (100 μg·ml−1) was co-incubated with the cells for 1, 3, 6, 12, and 24 hours, the cells were washed three times with PBS, detached by trypsin, and lastly collected by centrifugation at 1000 rpm for 5 min. The bottom cells were washed three times with PBS, and then the suspended cells were analyzed by BD LSR II Flow Cytometer equipped with BD FACSDiva (BD Biosciences) software.

### Treating stimulated inflammatory cells with NV, ZIF-8, and NV-ZIF

PBMCs were isolated from blood samples of two patients, one with AML and another with CLL, using a Ficoll gradient (Axis Shield, Norway). Cells were collected in complete RPMI 1640 medium (pH 6.5). PBMCs (106 cells) were then divided into five sets for each treatment and incubated with either ZIF-8 (5 μg·ml−1), NV (10 μg·ml−1), or NV-ZIF (5 μg·ml−1) for 1 hour. Cells were then stimulated with PHA (100 ng·ml−1) for 6 or 12 hours (the last 2 hours in the presence of brefeldin A). Intracellular cytokine staining was performed to determine the ability of CD8+ cells to express cytokines. The cells were surface stained with CD3+ APC (0.2 μg·μl−1; R&D Systems, Minneapolis, MN, USA). They were then fixed in 4% paraformaldehyde, resuspended in 0.25% saponin (S4521; Sigma-Aldrich, Germany), and stained with anti–IFN-γ PE-Cy7 [PE-Cy7 mouse anti-human IFN-γ (BD Biosciences), 0.2 μg·μl−1] and anti–TNF-α–PE-Cy7 [PE-Cy7 mouse anti-human TNF-α (BD Biosciences), 0.2 μg·μl−1] antibodies. Samples were analyzed using a BD LSR II flow cytometer equipped with BD FACSDiva (BD Biosciences) software.

### Animals and tumor models

All animal experiments were carried out in accordance with the Institute of Laboratory Animal Resources guidelines. Ethical approval was granted by the Institutional Animal Care and Use Committee of Zhejiang Academy of Medical Sciences, China.

Female BALB/c mice (4 weeks old, ~20-g body weight) were purchased from the Zhejiang Academy of Medical Sciences and maintained in a pathogen-free environment under controlled temperature (24°C). A total of 0.1 ml of 4T1 cells (5 × 105) was injected into the breast fat pad of the mice. The tumors were allowed to grow to ~100 mm3 before experimentation.

The tumor volume was calculated as (tumor length) × (tumor width)2/2.

For tumor accumulation studies, the mice were randomly divided into two groups (n = 3) and intravenously injected with XenoLight DiR–NV–ZIF or Rh–NV–ZIFMCF [corresponding to NV (3.0 mg·kg−1)]. The fluorescent images were obtained under an IVIS (CRi USA, IVIS: 710 excitation/760 emission). The tumor samples were then collected at the desired time after injection and were digested using concentrated nitric acid. The amount of Zn2+ in the tumors was measured using ICP-MS.

For antitumor activity study, 4T1 tumor-bearing mice were randomly divided into six groups (n = 5) and intravenously injected with (i) 200 μl of physiological saline, (ii) 200 μl of ZIF-8 solution, (iii) 200 μl of NV-ZIF solution, (iv) 200 μl of NV-ZIFMCF solution, (v) 200 μl of NV solution, and (vi) 200 μl of NVMCF solution, respectively. The dosage of NV is 3.0 mg·kg−1. Mice received treatment four times every 3 days. Physiological saline that is used for in vivo application is 1× PBS (0.01 M). Tumor volume and body weight were measured every 3 days. In the histological assay, the tumor tissues were fixed in 4% paraformaldehyde for 24 hours. The specimens were dehydrated in graded ethanol, embedded in paraffin, and cut into 5-mm-thick sections. The fixed sections were deparaffinized and hydrated according to a standard protocol and stained with H&E for microscopic observation. Tumor sections were also stained with antibody against Ki-67 (Abcam, USA) to visualize viable CCs.

Blood samples (0.1 ml) were taken from retro-orbital sinus to isolate serum for analysis, 48 hours after single injection. TNF-α (MTA00B; R&D systems) and IFN-γ (MIF00; R&D systems) were analyzed with enzyme-linked immunosorbent assay kits according to the vendors’ protocols.

To study the immune cells inside tumors, tumors were harvested from mice in different groups and cut into small pieces. After being ground by the rubber end of a syringe in cell strainers, tumor tissues were treated with 0.25% trypsin-EDTA solution for 5 min at 37°C. Then, cells were filtered through nylon mesh filters with a size of 70 μm and washed with PBS. The single-cell suspension was then incubated with anti-CD16/32 (BD Pharmingen; catalog: 553141) to reduce nonspecific binding to the fragment crystallizable region (Fc receptor). Cells were further stained with anti-mouse FOXP3 (eBioscience; catalog: 12-5773-82) antibodies according to the manufacturer’s protocols. Last, flow cytometry was used for cell sorting.

### Statistical analysis

Data are reported as means ± SD. The differences among groups were determined using one- or two-way analysis of variance (ANOVA) analysis. Statistical significance was calculated by one- or two-way ANOVA and Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Acknowledgments: We thank R. Langer, Institute Professor, MIT, for feedback and comments. We acknowledge H. Alrabiah, Associate Professor at the Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University and H. I. Aljohar, Assistant Professor at the Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University for providing nivolumab. Funding: This work was funded by the King Abdulaziz City for Science and Technology (KACST) through the MERS-CoV research grant program (number 20-0004), which is a part of the Targeted Research Program (TRP). Author contributions: N.M.K. conceived the idea. S.K.A. carried out synthesis and characterization of NV-ZIF and NV-ZIFMCF and in vitro studies. S.S.Q., S.S., A.A., R.H., W.B., M.A., MR.A., and J.M. helped in characterization and in vitro studies. Z.M. designed and performed in vivo studies. S.K.A. and N.M.K. designed the experiments and wrote the paper. All authors discussed the results and commented on the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

## Functionality of immune cells in early life — ScienceDaily

• January 22, 2021

A study shows that putatively immature dendritic cells found in young children are able to induce robust immune responses. The results could lead to improved vaccination protocols.

Dendritic cells are a vital component of the innate immune system, which constitutes the body’s first line of defense against infectious agents and tumor cells. Their job is to activate the T-cell arm of the adaptive immune system, which confers specific and long-lasting protection against bacterial and viral infections. Dendritic cells engulf and degrade proteins that signal the presence of invasive pathogens. The resulting fragments (antigens) are displayed on their surfaces. T cells bearing the appropriate receptors are then activated to seek out and eliminate the pathogen. Newborns and young children have fewer dendritic cells than adults, and these juvenile cells also carry fewer antigen-presenting complexes on their surfaces. Based on these observations, immunologists have generally assumed that these cells are functionally immature. However, new work published by a research team led by Professor Barbara Schraml at LMU’s Biomedical Center has shown — using the mouse as a model system — that this assumption is in fact erroneous. Although early dendritic cells differ in their characteristics from those of mature mice, they are nevertheless quite capable of triggering effective immune reactions. The new findings suggest ways of boosting the efficacy of vaccines for young children.

With the help of fluorescent tags attached to specific proteins of interest, Schraml and her colleagues traced the origins and biological properties of dendritic cells in newborn and juvenile mice, and compared them with those of mature animals. These studies revealed that dendritic cells are derived from different source populations, depending on the age of the animal considered. Those found in neonatal animals develop from precursor cells produced in the fetal liver. As the mice get older, these cells are progressively replaced by cells arising from myeloid precursors, a class of white blood cells that originates from the bone marrow. “However, our experiments demonstrate that — in contrast to the conventional view — a particular subtype of dendritic cells named cDC2 cells is able to activate T-cells and express pro-inflammatory cytokines in young animals,” Schraml explains. “In other words, very young mice can indeed trigger immune reactions.”

Nevertheless, early cDC2 cells differ in some respects from those found in adult mice. For example, they show age-dependent differences in the sets of genes they express. It turns out that these differences reflect the fact that the signaling molecules (‘cytokines’) to which dendritic cells respond change as the mice get older. “Among other things, the array of receptors that recognize substances which are specific to pathogens changes with age,” says Schraml. “Another surprise for us was that early dendritic cells activate one specific subtype of T-cells more effectively than others. Interestingly, this subtype has been implicated in the development of inflammatory reactions.”

The results of the study represent a substantial contribution to our understanding of the functions of dendritic cells, and they could have implications for medical immunology. The immune system of newborns differs from that of more mature individuals insofar as immune responses in early life tend to be weaker than those invoked later in life. “Our data suggest that it might be possible to enhance the efficacy of vaccinations in childhood by, for example, adapting the properties of the immunizing antigen to the specific capabilities of the juvenile dendritic cells,” says Schraml.

Story Source:

Materials provided by Ludwig-Maximilians-Universität München. Note: Content may be edited for style and length.

## 4 Best Testosterone Boosting Supplements – Top Natural Boosters for Increasing Testosterone Levels

• January 22, 2021

Testosterone is the main male hormone. It is an anabolic steroid that is responsible for the development of the male sex organs in the puberty stage. This hormone increases strength, muscle mass, and sex drive. It also decreases fat mass and gives an athletic body. While the levels of testosterone are the highest during early manhood and decrease with progressing age. So, many men have low testosterone levels and experience many health conditions like erectile dysfunction, loss of muscle mass, gain in fat mass, reduced energy, low sexual desire, and depression.

So, there is a need for extra testosterone in men that according to the medical experts can be obtained from testosterone supplements. Artificial steroids have many harmful effects and cause many health disorders. But some natural testosterone booster supplements have natural ingredients and can provide extra energy, sex drive, and an athletic body like the youthful days. There are two natural supplements available in the market now that can effectively boost up testosterone levels safely.

Why Should Men Use Testosterone Boosters?

With advancing age, the natural testosterone production in men decreases. The man gains fat mass and loses his muscle mass. He starts losing his sex drive and starts experiencing sexual disorders like erectile dysfunction and low stay power. Visiting clinics are costly and the medicines prescribed have long-term side effects. In contrast, some of the natural supplements that are available in the market to boost testosterone levels are safe, effective, and affordable. After research on the available testosterone booster supplements, the author has found the two best ones that are reviewed for the readers.

Top 4 Natural Testosterone Booster Supplements

Testosterone is a chief and important hormone in the body of a male. It’s crucial to have optimum production of the specified hormone in the human body if a person wishes to develop muscle mass. As men start aging, their body generates very little testosterone which destroys the overall structuring of the body. Their nature of sexual functions and life also gets diminished. This is why all people need a testosterone booster such as Testogen and Testo Max. These supplements are made up of all-natural high-quality ingredients. So, they don’t give artificial and synthetic testosterone to the human body such as steroids. The supplement facilitates the generation of the crucial and chief male hormone that provides the consumers with better recovery of muscles. The product also provides the user’s body with much-needed energy.

Firstly, this product review addresses the characteristics of the Testogen supplement, how it works, the components used in the product, and its advantages on the human body.

1. Testogen

• Its chief role is to develop the user’s muscle mass. It stimulates the optimum generation of testosterone in the human body.
• It gives the human body much-required energy to execute day-to-day tasks after going through an extreme workout session.
• The company of the Testogen supplement offers the users a 100-day money-back policy.

Testogen Natural and Strong Testosterone Booster

This is an organic testosterone booster that has become the best seller in a few years. It contains effective ingredients that can boost up testosterone production, increase libido, and muscle mass. It contains very powerful and effective natural ingredients that can detoxify the body and boost-up the testosterone production from inside.

Ingredients

• D-Aspartic Acid  – D-Aspartic Acid regulates amino acids that help to generate testosterone.
• Vitamin D3– Vitamin D3 increases testosterone levels in the body.
• Magnesium – Magnesium generates testosterone by enhancing the enzymes. This is required for the production of the hormone.
• Extract of Korean Red Ginseng – This component stimulates the libido which improves the user’s mood and gives him stronger and harder erections.
• Extract of Nettle Leaf  – Extract of Nettle Leaf helps to release testosterone from its beholden form. The sex hormone Binding Globulin is also released with testosterone to increase libido.
• Vitamin K1 – Vitamin K1 makes human bones healthier and stronger. Stronger bones can suck up Vitamin D in a better way. This increases testosterone levels.
• Extract of Fenugreek – This component helps in improving the overall strength and stamina of the user’s body. The seed of Fenugreek increases the testosterone levels of the body.
• Zinc – Zinc helps to increase the levels of testosterone in the human body naturally.
• Vitamin B6 – This Vitamin helps in growing androgens. Androgens are a very vital and crucial hormone. This increases the testosterone levels in the body. This hormone is extremely important in the male reproductive system.
• Bioperine – Bioperine helps in absorbing all the components present in the supplement effectively.
• Boron – Boron helps in identifying and treating erectile dysfunction. It improves both muscle and bone strength.

More Information on Testogen Can Be Found On The Official Website Here

How to Use Testogen?

While steroidal injections pump in artificial testosterone hormone in the body, Testogen contains natural ingredients that boost up the natural production of testosterone. To balance the testosterone levels in the body, the company recommends 4 capsules daily with a glass of water with breakfast. The company claims that if taken with breakfast, the user will be full of energy the whole day and work out hard in the gym.

How Does the Testogen Work?

Testogen is manufactured with all-natural ingredients. This makes it the best selling product. These components present in the product safely enhance the levels of testosterone in the body. This does not contain any synthetic ingredients or products that can harm the body. Testogen enhances the whole system of the human body and stimulates the body to generate more and more testosterone in the body. People who have low testosterone levels in the body can use this product. This supplement balances the hormone levels in the body safely. According to the manufacturers of Testogen, regularity in consuming the supplement will give faster favorable results.

Pros

• Improves your testosterone levelsThe supplement improves the hormone levels in the body. The combination of all the ingredients would boost the production of testosterone in the body. This will increase the testosterone levels in the body.
• Contains all-natural ingredients The product has 100% all-natural ingredients that work effectively and helps the human body in numerous ways. This product does not have chemicals or artificial additives. This helps in keeping the body healthy and active. This provides the user with all the benefits naturally.
• Helps in building musclesIt gives the user’s lean muscles that the user wanted to get. It will provide the customer with these benefits without even going to the gym. The supplement helps to build lean muscles.
• Helps lose stubborn fatTestogen enhances the levels of testosterone in the body. This helps in improving the metabolic system of the body. It helps the user in losing stubborn and accumulated excess fats in the body in any troublesome areas.
• Improves the moodLow testosterone levels in the body affect a person’s mood drastically. A bad mood gets any person irritated very quickly. Testogen improves the user’s mood and changes the user’s bad mood into a delightful one.
• Increases the libido – People having problems with their sex drive and sex life can use Testogen. This will help them increase their self-confidence and enhance libido.
• Easy useThe supplement is in pill formulation. So, it is very easy to consume compared to the other testosterone-boosting supplements in the entire market.
• Fast resultsThe user will be able to see noticeable results in just 14 days of starting to consume the supplement. The user can see visible results if the user takes the suggested dosage regularly.
• No side effects The supplement is manufactured by using all-organic components. So, the supplement does not have any side effects. This supplement does not harm the body in any way.

Cons

• Only available on their websiteThe user can buy the supplement from their authentic, official website. They offer worldwide user shipping. If the user wants to get the best deals, the user will have to purchase it from the website.
• Strict dosage The user should follow the suggested dosage to have the desired results.

Reasons to Purchase Testogen

The user should purchase this supplement to get benefits in both the overall health and hormone levels. Testogen is made up of high-quality all-natural ingredients. This ingredient naturally boosts the production of the hormones and enhances testosterone levels. The supplement also contains potent ingredients that reduce symptoms of joint discomfort and boosts the immune system.

2. Testo Max

• The supplement is an organic formulation that works effectively for young, middle-aged, and old males.
• It helps males to develop muscle mass. This also helps in getting stronger.
• The product helps in melting accumulated extra fat from the human body, particularly the belly fat.
• Testo Max helps in controlling blood pressure. It also helps in overcoming fatigue.
• The developer affirms that Testo Max can enhance testosterone levels by more than 40% in less than 2 weeks of starting to use the product in regular dosage.

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Testo Max The Safe Natural and Effective Testosterone Booster

Muscle building becomes difficult as natural testosterone levels decrease with age. So, with hours of work out in the gym also, men cannot gain muscle mass. Here, supplementation with a natural and safe dietary supplement called Testo Max will naturally boost testosterone levels. Men take this supplement to easily sustain the workout and get an increased muscle mass and lean fat-free body. The seller claims that men who have erectile dysfunction and low-libido can benefit hugely from this natural testosterone booster.

Ingredients

Testo Max has 12 natural ingredients that are effective in boosting the natural production of testosterone.

• Vitamin B6 – The human body doesn’t have only the hormones estrogen and testosterone. It also has a very important androgen, a precursor that helps boost the blood’s T-hormone level. Vitamin B6 enhances androgen levels in the body by instructing the body to boost up production. This Vitamin also improves the mood that is required by men suffering from low testosterone levels that gives rise to depression and mood swings.
• Vitamin D3 – This is an essential vitamin found in sunlight, but many are deficient in it. Research proves that men who are deficient in Vitamin D have hormone imbalances, including testosterone deficiency. So the manufacturers have included a big boost of this useful Vitamin so that the testosterone levels remain healthy.
• Vitamin K2 – Vitamin K2 is an important constituent of this supplement that balances all the body hormones. It signals the brain to increase testosterone production so that sex drives increase. It also helps in removing ED or erectile dysfunction. in
• D-Aspartic Acid – This is an important amino acid that acts as a neurotransmitter. It boosts the testosterone level, increases sperm count, and improves fertility.
• Zinc – This mineral regulates the functioning of the pituitary gland so that healthy levels of the male hormone testosterone is secreted. It also improves immunity and prevents the onset of age-related diseases.
• Magnesium – It reduces the SHGB count in the blood which is responsible for low absorption of testosterone hormone. By reducing the SHGB in the blood, magnesium increases the free flow and absorption of the testosterone hormone. This also gives the body a boost of energy to sustain rigorous bodybuilding exercises.
• Ginseng – Medical use of Ginseng has been done from ancient days. This is a powerful antioxidant that improves the immunity and overall lifespan of the user. It improves all bodily functions and also prevents diseases like erectile dysfunction and low libido.
• Mucuna Pruriens – The main reason for lower T-levels is high estrogen in the bloodstream. Mucuna Pruriens is a source of levodopa, an amino acid boost testosterone level improves the addition of muscle mass to give lean muscles easily. It also decreases the accumulated fat in the body and gives a slim and manly body structure.
• Luteolin – It is a potent aromatase inhibitor. This works on the aromatase, and also reduces the excess estrogen from the blood. It balances estrogen and testosterone levels perfectly so that the body gets to use the male hormone. Even in men above 50, this component gives an enhanced sex drive, high energy levels, and a youthful lean body.
• Nettle Root – It has many health benefits, so the formulators have included it in Testo Max. This root extract allows the free availability of testosterone in the blood.
• Boron – This is known to have a positive effect on the working of testosterone. It improves the functionality of the male hormone, reduces inflammations and joint pains by nurturing healthy bones and cartilages.
• BioPerine – Bioperine is a very important component that allows the easy processing of nutrients. The formulators use it to improve the effectiveness of Testo Max. It can also improve protein levels and lean muscle formation.

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How to Use Testo Max

The manufacturers advise the users to consume 4 capsules daily with a glass of water with the morning meal or before the exercise schedule to get an extra boost of energy.

How Does Testo Max Work?

This supplement boosts testosterone production from inside the body by improving the functioning of the pituitary gland. It also contains ingredients that increase energy levels, sex drive, immunity, and muscle mass. It also

Pros

• It gives very high energy levels throughout the day.
• It gives more strength to the body for sustaining more bodybuilding activities.
• Improves libido and cures erectile dysfunction.
• Makes the user happy and removes anxiety and depression
• Improves immunity and prevents diseases like prostate cancer.

Cons

• The effect of this natural supplement is fast but not miraculous like a steroidal injection.

The users should buy Testo Max to boost energy levels, increase muscle mass, and improve the sexual drive. Available steroids boost the testosterone levels very fast but have side effects like physical and psychological changes and lead to prostate cancer. Testo Max is a natural alternative that improves immunity, muscle mass, aids fat-loss, improves libido, and joint health. Even older men benefit hugely from Testo Max as it enhances performance and reduces recovery time.

3. Testofuel

• Its main role is to help build muscle mass in men by facilitating the optimal production of testosterone in the body.
• It also provides the body with much-needed energy to perform day-to-day activities after an intense workout session.
• The company offers a 90-day refund guarantee.

Testofuel is one of the most effective muscle-building supplements available in the market. It has emerged to become the most trusted supplement in its industry. Users across America have been getting positive results from using the product as it helped them gain muscle mass. Men have been experiencing results from supplementing Testo fuel with their diet that indeed gives them the required energy to carry out an intense gym session and allows better muscle recovery.

As per the creators, it’s one of the safest supplements in the market. They have only used pure and potent substances in the composition. All the constituents are plant-based and work in sync to increase the production of testosterone in a male’s body.

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Listed below are the main ingredients used in the composition of Testofuel:-

• Fenugreek: It’s a naturally occurring herb found in the Mediterranean conditions. It has a sweet aroma and is used in Asian cuisine. It’s rich in zinc, selenium, and magnesium, three of the micronutrients most people are deficient in. The herb helps inhibit the sex hormone-binding globulin (SHBG) which is responsible for taking up the most amount of the testosterone produced. And, as the herb puts brakes on this hormone, it becomes easier for the “free” testosterone to work.
• D-Aspartic Acid: It’s a significant amino acid that aids in the regulation of testosterone synthesis, helps in building muscle mass, and the production of growth hormone. A recent study says that men who consumed a regular dose of the supplement increased their testosterone levels by 8-10%
• Vitamin D: This micronutrient is extremely important for male hormones and so it’s named the sixth steroid. Even though it’s obtained from the UV rays, it’s scarce for men living in the West. This is where the supplement helps. The micronutrient can optimize testosterone production in the body.
• Asian Red Panax Ginseng: It’s an Asian herb that is known to improve mood patterns and improve cognitive abilities. It also helps in building the immune system of the body. It’s a libido enhancer and has direct impacts on testosterone levels.
• Extract of Oyster: It’s an ancient food used to enhance libido in the body. It’s an aphrodisiac that has high-zinc content and can easily elevate the testosterone levels in the body.
• Vitamin B6: “B” vitamins are probably the most important category of vitamins there is. Vitamin B6 is also called pyridoxine and it promotes physical and mental well-being. This Vitamin is known to reduce the production of the primary female hormone estrogen and boost testosterone production.

There are other ingredients as well like vitamin K2, Magnesium, and Zinc. Readers can find the entire list of ingredients on the official website of the company.

How Does Testofuel Work?

As per the website of the manufacturer, they have used pure and potent forms of substances that are naturally occurring in nature. They are tested so that the constituents can work in sync to increase the production of testosterone in the body. The creators emphasize that the supplement doesn’t provide testosterone artificially to the body, like some steroids, but helps optimize its production in the body. It’s a known fact that if someone wants to bulk, their body’s production of testosterone must be optimal. More production of testosterone induces better post-workout recovery and also provides energy.

Dosage

One bottle of Testofuel contains 120 pills and each bottle is a one-month supply, so according to that, users must take 4 pills every day.

Pros

• It helps users to develop muscle mass.
• The supplement enhances the production of testosterone in the body.
• It gives the body more energy to push through an intense workout session.
• Testofuel promotes better muscle recovery.
• It improves the user’s mood and cognitive abilities.
• The supplement can also boost the immune system of the body.
• There’s a 90-day refund guarantee backing the product.

Cons

Where to Buy the Supplement and What are the Available Packages?

Users can visit the official website of the seller and see the available packages. They can order the package that’s most suited to their requirements. One box of Testo fuel is available for $65, two boxes$130, and three for $195. The company offers free shipping for orders across the US and the UK. They have also backed the product with a 90-day money-back guarantee, so customers can return the supplement if they want, and get a refund. Click here to Get the Best Deal on TestoFuel from the Official Website 4. Prime Male • It’s a natural formula that works for young and old men. • It helps men build muscle mass and get stronger. • The supplement helps shed excess fat from the body, especially from the belly region. • Prime Male helps control blood pressure levels and helps overcome fatigue. • The seller claims that it can boost testosterone levels by 42% in only 12 days of consuming a regular dose of the supplement. Prime Male is one of the most popular muscle-building supplements available in the market. It’s formulated by Roar Ambition Ltd., a trusted supplement company based in the UK. Just like all their other supplements, the creators have used natural substances to formulate the product. They claim that the proprietary blend used by them is ideal for mature men. As per the website of the manufacturer, the supplement will help boost testosterone production in the body. It’s made with an all-natural formulation under the supervision of experts and doctors. Each ingredient is tried and tested for purity and potency before being added to the composition. Given below are the major ingredients used in the composition of the supplement- • D-Aspartic Acid: An important amino acid, D-Aspartic acid helps in facilitating muscle growth. It also helps regulate testosterone in the body and use it for carrying out important activities. It’s a safe substance that boosts the production of testosterone by 12% in the short-term. • Zinc: Most men are unaware that they are probably deficient in this micronutrient. This is because to optimize testosterone production in the body, zinc is absolutely necessary. And, if people want to get the necessary amount of zinc from foods, they would have to eat a lot. This supplement, therefore, helps users cover the daily intake and optimizes testosterone production. • Vitamin D3: Another micronutrient that helps regulate testosterone production in the body, vitamin D3 intake is quite important. Everyone knows that the sun is the primary source of vitamin D3 but the amount obtained isn’t adequate and the supplement addresses it. • Vitamin K2: A micronutrient known to work simultaneously with vitamin D3. It helps vitamin D3 get absorbed quickly and also optimizes ‘T’ levels in the body. The creators use Menaquinone-7 in the composition of the supplement which happens to be an enhanced version of vitamin K2. • Ashwagandha Extract: It’s an ancient herb used to treat sexual problems. It’s also known to alleviate tension and stress from the mind which helps in improving the sexual performance of the user. Many studies indicate that it can enhance testosterone levels as it reduces anxiety but more importantly, ashwagandha gives energy to the user for an intense workout session. • Luteolin: as much as optimizing the production of testosterone is important, reducing the build-up of the primary female hormone, estrogen, is just as necessary. Luteolin does just that, inhibiting the production of estrogen. • Boron: Boron helps in maintaining the balance between hormones in the body. It can reduce inflammation and improve bone health in the body. Boron inhibits the growth of sexual hormone-binding globulin (SHBG), a hormone that needs to be stopped to increase testosterone production. ==> Read More Here: Don’t Miss Out Today’s Special Offer <== How Does Prime Male Work? There’s a simple working for every testosterone booster out there in the market. First, the ingredients used in the supplement need to get absorbed in the bloodstream of the individual. When it gets assimilated, Prime Male works to optimize the production of testosterone in the body. Greater testosterone levels ensure better recovery of muscles. And, it facilitates an increase in muscle mass. The supplement also contains ingredients that are known to increase the energy levels in the body, which aid people to push through an intense workout session. How to Use Prime Male? Every Prime Male bottle contains 120 pills of the testosterone-boosting supplement. For the best results, users must take 4 capsules of the supplement every day. Users may take 2 pills before their workout session and 2 at night before going to sleep. Pros • The company claims that they have used naturally occurring substances in the composition of the supplement to formulate it. • It can boost testosterone production in the body by 40-45% in only a few weeks of using it. • The supplement helps in better muscle recovery, thereby aiding muscle growth. • The seller offers free shipping for all orders from the UK and the US. • It provides extra energy to the users for them to carry out intense workouts in the gym. Cons • The supplement can be purchased only from the official website of the company. Pricing Policy There are four packages of the supplement available on the company’s website. Users can visit the website and choose the most suitable package for themselves. The one-month package is available for$65, two-month supply for $130, four-month supply for$195 (one bottle is complimentary), and six-month package for \$260 (two bottles complimentary).

Symptoms of Low Testosterone

Any person can understand if their testosterone levels are low if their body shows any of the following signs:

1. Fat Accumulation– Accumulation of fat despite exercising and following a good diet.
2. Decreased Muscle Mass– Although muscle mass decreases with age, if it happens at an alarming rate despite exercising and following a good diet, the reason can be low testosterone levels.
3. Mood Swings– The reason for mood swings is associated with change in hormonal levels.
4. Decreased Libido– Testosterone is the sex hormone in males, the lack of which results in decreased libido.

Working of Testosterone Boosters

The work of testosterone boosters is to help the body produce more testosterone. They lower the levels of estrogen and prolactin and thus, help in the release of testosterone. All this is possible with the help of special ingredients that are combined together in specific ratios to make an effective formula.